inova news 8 edição

8/16/2019 INOVA News 8 Edição

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First described in 1982, antineutrophil cytoplasmatic antibodies (ANCA) representa diagnostically important group of autoantibodies. Indirect immunofluorescence

(IIF) on ethanol fixed human neutrophils was not only the first method described for

the detection of ANCA, it is still proclaimed as the method of choice for the first line

screening of sera from patients suspected to suffer from ANCA associated vasculitis

(AAV). The labor-intensive methodology inherent to IIF prompted new technological

advancements such as automated digital image analysis. Such technology allows for

standardization, reduction of hands-on time, and facilitation of case review.

While low sensitivity and specificity of early enzyme immunoassays (ELISAs) caused

much controversy, recent advances, such as the development of second and third

generation capture ELISAs offered significant improvements in diagnostic accuracy.

Ongoing innovations in the field of antigen specific solid phase immunoassays have

continuously improved the analytical and clinical performance characteristics of these

assays, supporting both diagnosis and follow-up. The recently developed chemilumi-

nescent immunoassays (CIA) offer results in 30 minutes while allowing for high

sensitivity and specificity. These advances have helped to facilitate new opportunities

in the formerly unresolved associations of ANCA, such as the specific detection of

PR3-ANCA in ulcerative colitis.

This issue of the INOVA Newsletter covers historical perspectives from thirty years ago

to the current advances available today.

I hope you will enjoy reading!

Antineutrophil Cytoplasmic Antibodies:Historical perspectives and new advances

IN THIS ISSUE

INOVA NEWS

No. 8p2 An introduction to ANCA-associatedvasculitis | Anto nell a Ra dice, PhD

p5 Diagnostic methods for ANCA: past and p Joh anne s Sc hulte -Pelku m, P hD

p9 ANCA testing in emergency setting |Lucile Musset, PhD; Makoto Miyara, MD, PhD

p12 A new look at ANCA with NOVA VIEW®Gabriella Lakos, MD, PhD; Carol Buchner, MT (ASCP)

p16 Anti-PR3 antibodies in ulcerative colit Mich ael Mahl er, Ph D; Ga ry L. N orm an, PhD;

Marco s Lo pez- Hoyo s, MD, PhD

Johan nes S chulte -Pelk um, Ph DDirector of Research and Development

INOVA Diagnostics, Inc.


2/202 | INOVA NEWS No. 8

An introduction to

ANCA-associatedvasculitis

Antonella Radice, PhD

Laboratory Manager - Immunology,

Autoimmunity and Microbiology

San Carlo Borromeo Hospital

Milan, Italy

Discovery of ANCA

Antibodies to neutrophil cytoplasmic components(ANCA) were first described in 1982 by Davies et al. insome patients with necrotizing glomerulonephritis(FNGN) and symptoms of systemic vasculitis.1 In 1985

van der Woude et al. reported the strong association ofANCA producing a diffuse granular cytoplasmic stain-ing pattern (cANCA) on ethanol-fixed neutrophils andgranulomatosis with polyangiitis (formerly Wegener’sgranulomatosis (WG)); a few years later, ANCA, produc-ing a perinuclear fluoroscopic pattern (pANCA) on thesame cellular substrate were described in patients withidiopathic necrotizing crescentic glomerulonephritis(iNCGN) and microscopic polyangiitis (MPA).1 (Figure 1a.and Figure 1b.)

The method available at that time to detect the cANCA

and pANCA pattern was the indirect immunofluores-cence test (IIF) on normal human ethanol-fixed leuko-cytes.2 Over 30 years since its first use, IIF remains thedesired method to screen samples in patients suspect-ed of vasculitis.

Function of target antigens-PR3 and MPO

The target antigens of ANCA are located in theprimary granules of neutrophils and have antibacte-

rial properties. PR3 and MPO are recognized in mostANCA-positive small vessell vasculitidies. PR3 is aweak cationic protein consisting of 228 amino acidresidues (MW 29-30 kD), belonging to the trypsin familyof serine proteases. PR3 is synthesized as a prepro-enzmye and subsequently processed in four stepsinto the mature form. It is stored in the azurophilicgranules of neutrophils, but can also be found withinthe membrane of secretory vesicles, and also expressedat the plasma membrane. PR3 is physiologically inhib-ited by α1-antitrypsin.4 PR3 disintegrates tissue to allowthe passage of neutrophils into an inflammatory focus

and is also involved in neutrophil maturation. MPO isa 150 kD heterdimer peroxidase enzyme abundantlyexpressed in neutrophils. The enzyme is characterizedby a powerful bactericidal activity, whose peroxidaseactivity is physiologically inhibited by ceruloplasmin.5

The identification and purification of PR3 and MPOantigens resulted in development of several immunoas-says for the quantitative detection of antibodies specific foPR3 and MPO; the conventional enzyme-linked immuno-sorbent assays (ELISAs) and, more recently, methodsbased on different fully automated technologies.3

a.)

b.)

Figure 1a.) cANCA is largely due to the presence of autoanti-bodies targeting the serine protease proteinase-3 (PR3-ANCA).Figure 1b.) pANCA is caused by antibodies directed against manyantigens, among which myeloperoxidase (MPO-ANCA) is themost frequent in primary systemic vasculitis.3


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Pathogenesis of ANCA disease

Numerous in vitro studies demonstrate that bothMPO-ANCA and PR3-ANCA are capable of activat-

ing neutrophils and monocytes through Fab’2 and Fcengagement, which initiates several signal transduc-tion pathways (Figure 2).6 Activation of these leuko-cytes results in adhesion to endothelial cells, causingendothelial damage. Studies in animal models provideconclusive evidence that anti-MPO antibodies inducenecrotizing and crescentic glomerulonephritis andsystemic small vessel vasculitis. Anti-MPO antibodies inthe absence of functional T cells are capable of causingglomerulonephritis and vasculitis, and the inductionof this disease is dependent on neutrophils.6 This canbe exacerbated by a variety of cytokines, is dependent

on activation of the alternative pathway of comple-ment, and is abrogated by inhibition of the alternativepathway and by anti-C5 receptor antibodies. 6 Thesestudies provide a basis for exploring novel therapeuticstrategies.

Figure 2. Pathogenesis of ANCA-associated vasculitis

Neutrophil

PrimingChemotaxis

Endothelium

ANCA antigen

CD11b

Endothelial adhesionmolecules

Cytokine recptor

Fc receptor

Cytokine/chemokine

C5a receptor

C5a

C5a, chemokines

In situ ICXformation +AP Complementactivation

O2O2

Infection ANCA

Release ofproinflammatorycytokines

ANCA classification

ANCA are the serological hallmarks of idiopathic systemicvasculitis, and the term ANCA-associated vasculitis (AAV)

has been used to collectively name those primary smallvessel vasculitic syndromes in which circulating ANCA arecommonly found (microscopic polyangiitis and its renallimited form, granulomatosis with polyangiitis, Churg-Strauss syndrome).7 This approach, adopted by theChapel Hill International Consensus Conference (CHCC)and by the European Vasculitis Study Group (EUVAS), issupported by the striking clinical and histological similar-ities between the AAV, the widespread use of ANCA asa diagnostic marker, and the growing evidence of theirpathogenetic potential.8

Names of the common forms of vasculitis have beenrecently revised so that the eponyms such as WG andCSS have been changed with granulomatosis withpolyangiitis (GPA) and eosinophilic granulomatosiswith polyangiitis (EGPA), respectively.9 The new diseasenomenclature system adopted by the 2012 CHCCfor defining small vessel vasculitis is listed in Figure 3.

After the standardization of the methods for ANCAdetection and the evaluation of their clinical applicationwas agreed upon, a document produced by an interna-tional consensus of experts was published, with sugges-tions for the correct ANCA testing and reporting.10

The Chapel Hill Consensus Conference

nomenclature defines 10 primaryvasculitides based on vessel size.2

Giant cell arteritis Takayasu arteritis

Polyarteritis nodosaKawasaki disease

Henoch-Schonlein purpuraCryoglobulinemic vasculitis

Anti-GBM

Large- tomedium-sizedartery

Small-sizedartery

Arteriole

Capilary

Venule

Vein

Aorta

Leukocytoclasticvasculitis

Microscopicpolyangitis

Granulomatosiswith polyangitis

ANCAassociated

vasculitisChurg-Strauss

Figure 3. The Chapel Hill Consensus nomenclature defines 10

primary vasculitides based mainly on vessel size

Adapted from Falk, R and Jennette, J. 2010 J Am Soc Nephrol


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An introduction to ANCA-associated vasculitis...Continued

4 | INOVA NEWS No. 8

Table 1. Indications for ANCA testing

Clinical indication for ANCA testing

Glomerulonephritis, especially rapidly progressiveglomerulonephritis

Pulmonary hemorrage

Cutaneous vasculitis

Multiple lung nodules

Chronic destructive disease of the upper airways

Long standing sinusitis or otitis

Subglottic tracheal stenosis

Peripheral neuropathy

Retro-orbital mass

Other possible indications for ANCA testing

Pulmonary fibrosis, with systemic features

Episcleritis, uveitis, retinal vasculitis, with systemic features

Table 2. Summary of the international consensus statement ontesting of ANCA

Minimum requirementsPerform IIF on all sera; 10% of ANCA-positive sera aredetected only by IIF

Samples with IIF positivity should immediately be tested forboth PR3 and MPO ELISA

Optimal requirements

IIF titration should be performed for sera positive only by IIF

The inclusion of the most recent positive serum in the IIF orELISA studies may be useful in demonstrating a change inantibody level

ANCA testing

The International Consensus Statement on Testingand Reporting of ANCA was created to enhance the

diagnostic usefulness of ANCA testing in patientssuspected of having vasculitis. Testing for ANCA in thepresence of certain clinical indications (Table 1) yieldsa high positive predictive value. Guidelines stipulatetesting samples with IIF on ethanol fixed leukocytes (orpurified neutrophils) and PR3/MPO by solid phase assaysuch as ELISA (Table 2). Testing all IIF positive samplesby ELISA produces sensitivities of 73% and 67% for GPAand MPA, respectively, and a diagnostic specificity of99%.9

References

1. Radice A, Sinico RA Antineutrophil cytoplasmic antibodies (ANCA).Autoimmunity 2005; 38(1):93-103.

2. Wiik A. Delineation of a standard procedure for indirectimmunofluorescence detection of ANCA. APMIS 1989; 6:12–13.

3. Radice A, Bianchi L, Sinico RA. Anti-neutrophil cytoplasmicautoantibodies: methodological aspects and clinical significance insystemic vasculitis.Autoimmunity Rew 2013; 12(4):487-495

4. Radice A, Sabadini E, Sinico RA. Antineutrophil cytoplasmicautoantibodies with specificity for proteinase 3. In: Y. Shoenfeld, M.E.Gershwin, P.L. Meroni (Eds), Autoantibodies 2007 (2nd ed), Elsevier B.V;14:105–110.

5. Kallenberg CGM. Antineutrophil cytoplasmic autoantibodies withspecificity for myeloperoxidase. In: Y. Shoenfeld, M.E. Gershwin, P.L.Meroni (Eds), Autoantibodies 2007 (2nd ed), Elsevier B.V; 14:95-103.

6. Falk, R, Jennette JC. ANCA disease: where is this field heading? J Am SocNephrol 2010; 5:745-752.

7. Seo P, Stone JH.The Antineutrophil Cytoplasmic Antibody-AssociatedVasculitides. Am J Med 2004; 117:39-50.

8. Jennette JC, Falk RJ, Hu P, Xiao H. Pathogenesis of antineutrophilcytoplasmic autoantibody-associated small-vessel vasculitis. Annu RevPathol. 2013 Jan 24;8:139-60.

9. 2012 Revised International Chapel Hill Consensus ConferenceNomenclature of Vasculitides. Jennette JC, Falk RJ, Bacon PA , Basu N ,Cid MC, Ferrario MC et al. Arthritis & Rheum 2013; 65(1):1-11.

10. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ et al. InternationaConsensus Statement on Testing and Reporting of AntineutrophilCytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999; 111:507–513.

IN CONCLUSIONIn conclusion :

• ANCA are the serological hallmarks of idiopath-ic systemic vasculitis, and the term AAV has beenused to collectively name those primarly smallvessel vasculitic syndromes in which circulatingANCA are commonly found.

• Names of the common forms of vasculitis havebeen recently revised so that the eponyms suchas Wegener’s granulamatosis and Churg Strausssyndrome have been changed with granulomato-sis with polyangiitis (GPA) and eosinophilic granu-

lomatosis with polyangiitis (EGPA), respectively.

• The International Consensus Statement on Testing and Reporting of ANCA recommendsscreening by ANCA by IIF and to confirm anypositivity by both PR3 and MPO ELISA.


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Diagnostic

methods forANCA: past andpresent Johannes Schulte-Pelkum, PhD

Director of Research and Development - Immunopathology

INOVA Diagnostics

San Diego, CA

Introduction

Antineutrophil cytoplasmic antibodies (ANCA) are oneof the most common causes of small vessel vasculitisand are diagnostic hallmarks of microscopic polyangi-itis (MPA), Wegeners Granulomatosis1, more recentlyreferred to as granulomatosis with polyangiitis (GPA)2 and Churg Strauss syndrome, now called eosinophilicgranulomatosis with polyangiitis (EGPA).

ANCA were first reported in 1982 by Davies et al.3 andinvestigated to a greater extent in 1985 by van derWoude et al.4 Myeloperoxidase (MPO) and protein-ase 3 (PR3) were soon discovered as major antigenictargets for ANCA in neutrophils. After these antigenswere described,5, 6 continual developments were madeto improve associated diagnostic tests. The impor-tance of serological testing cannot be underestimatedin patients suffering from vasculitis. These individualsmust be swiftly diagnosed and proper therapy must beadministered to avoid renal failure.7, 8

The initial screening method for these autoantibodies is,according to the “International Consensus Statement on Testing and Reporting of ANCA”, IIF on fixed neutrophils ofhuman origin. The result of an initial IIF screening is verifiedby ELISA.9 In the past, second and third generation testsoften did not reach the sensitivity and specificity of IIF onethanol or acetone fixed neutrophils,10, 11 but new methodssuch as lateral flow and multiplex assays are now availablewith increased sensitivity and specificity.12, 13 The majorcontribution for the improved performance was the resultof different immobilization strageties culminating in betterexposure of relevant epitopes.

The requirement of sample batching for ELISA has nowdriven innovation to random access test systems withreduction in assay times. Systems combining randomaccess and chemiluminiscent immunoassay (CIA)

technology have been developed and offerdrastically reduced assay times of just 30 minutes.14

First generation ELISA Tests

After the target proteins for pANCA and cANCA weredescribed,5, 6 the first ELISA was developed usingpurified native PR3 and MPO antigens of various origin. These assays used simple direct coating methods andthe purity of the antigens often was not very high.Most of all the tests lacked comparability of results anddid not show strong correlation to IIF methods.10 This

lack of sensitivity was the result of different epitopeson the PR3 antigen being blocked by the directbinding process. The lack of standardization led to theInternational Consensus Statement on Testing andReporting of ANCA by a combination of ELISA and IIFtest methods.9

Second and third generation tests

Second generation ANCA tests utilize a capture ligand,generally consisting of a monoclonal antibody direct-ed towards a specific epitope. This method allows theprotein to retain its three dimensional structure15 whileproviding significantly higher sensitivity comparedto first generation tests. Protein modification such asbiotinylation led to third generation ELISAs, also aimedto prevent protein distortion. Spacer molecules areattached to PR3 antigen16 and the complex binds to thesurface of the ELISA plate by streptavidin coupling. Thismethod further increased the sensitivity and specificityand showed improved correlation with IIF.17

Other ELISA methods

An alternative approach to achieve sensitivity compa-

rable to IIF is a direct coated ELISA using native purifiedPR3 in mixture with a recombinant human PR3 antigen. This method purportedly offers significantly highersensitivity compared to first generation direct coatedELISA tests and second generation capture ELISA. Apromising performance analysis of this method waspublished by Damoiseaux et al. in 2009.18 However,a recent study presented at the 16th internation-al Vasculitis & ANCA Workshop in Paris did not showa significantly higher diagnostic performance of thisapproach.19

TESTING FOR ANTINUCLEAR ANTIBODIES | 5


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Lateral flow and multiplex assays

Historically, lateral flow assays (LFA) were regarded as fast,but lacking sensitivity compared to ELISA tests.20-22 The

development of liquid antigens for LFA has significantlyimproved their diagnostic performance. The sensitivity ofLFA is comparable to that of third generation ELISA testswith a result time of 20 minutes.12,13 Since LFA is basedon a universal IgG with the antigens added in solution,this test system can accommodate multiple analyt-es. Presenting the antigen in the liquid phase preventspotential epitope masking, as seen in some solidphase assays.

Multiplex technology allows for the detection of multi-ple antibodies in a single assay run. The antigen antibody

reaction is visualized using fluorescent dyes and is readby two lasers, one identifying the bead and the secondexciting the fluorophore coupled to the conjugate. Thistechnology allows detection of up to 100 different analyt-es in only one reaction. For the diagnosis of ANCA, testsare available which simultaneously measure antibodiesagainst PR3 and MPO. The sensitivity for PR3 is compa-rable to first generation solid phase ELISA, when a cut offsetting optimized for 100% specificity is used.23

BIO-FLASH®- Rapid ResponseChemiluminescent Analyzer

The QUANTA Flash® PR3 and MPO assays (INOVADiagnostics San Diego, CA) are novel CIAs performedon the BIO-FLASH® instrument using native purifiedantigens from human neutrophils (Figure 1). The CIAsare designed for the fully automated BIO-FLASH instru-ment, containing a luminometer as signal detector,as well as all the hardware and liquid handling acces-sories necessary to perform the assay. Native PR3purified from human neutrophils is covalently boundto paramagnetic particles. Native MPO purified fromhuman neutrophils is bound to paramagnetic particles

via an anchor molecule to improve immunoreactivity.

Patient serum is prediluted by the BIO-FLASH instru-ment with sample buffer in a small disposable plasticcuvette. Small amounts of the diluted patient serum,the beads, and the assay buffer are all combined intoa second cuvette, mixed, and then incubated for 9.5minutes at 37 °C. The magnetized beads are sedimentedand washed several times followed by addition of isolu-minol conjugated antibody, and again incubated for 9.5minutes at 37 °C. The magnetized beads are sediment-

ed and washed repeatedly. The isoluminol conjugateis oxidized when sodium hydroxide and peroxidesolutions (“Triggers”) are added to the cuvette, and theflash of light produced from this reaction is measured as

relative light units (RLUs) by the BIO-FLASH optical system.

The RLUs are proportional to the amount of isolumi-nol conjugate that is bound to the human IgG, whichis in turn proportional to the amount of autoantibod-ies bound to the antigen on the beads. Based on theresults of running two calibrators, an instrument specif-ic Working Curve is created, which is used to calculatechemiluminescent units (CUs) for each sample.

Diagnostic methods for ANCA: past and present...Continued


Figure 1. a.) Principle of the novel chemiluminescent immunoas-says.14 Paramagnetic beads are coupled with native PR3 or MPO. The beads are then incubated with diluted patient samples. After9.5 min incubation unbound antibodies are removed by washingAnti-human IgG isoluminol conjugate (Tracer) is added andbinds immobilized antibodies. After another 9.5 min incubation

unbound Tracer is removed by washing. b.) Trigger 1 and Trigger2 are injected and emerging light is measured. After injection of Trigger 1 and Trigger 2, the luminescence is measured as relativeluminescence units (RLU).

b.)

a.)



Kappa = 0.92 QUANTA Lite PR3 Percent agreement (95% confidence)

Positive Negative Total

QUANTA Flash®PR3 CIA

Positive 31 3* 34 Pos agreement 100% (88.8-100.0%)Negative 0 37 37 Neg agreement 92.5% (79.6-98.4%)

Total 31 40 71 Total agreement 95.8% (88.1-99.1%)

*all samples were from GPA patients

Kappa = 0.83 QUANTA Lite MPO Percent agreement (95% confidence)


QUANTA Flash®MPO CIA

Positive 29 6* 35 Pos agreement 100% (88.1-100.0%)

Negative 0 36 36 Neg agreement 85.7% (71.5-94.6%)


*3 samples from MPA and 3 from GPA patients

Table 1. Agreement between QUANTA Lite ELISA and QUANTA Flash CIA

Kappa = 0.83 cANCA Percent agreement (95% confidence)


QUANTA Flash®PR3 CIA

Positive 28 6* 34 Pos agreement 100% (87.7-100.0%)

Negative 0 37 37 Neg agreement 86.6% (72.1-94.7%)


* 5 samples from GPA and 1 from MPA patient

Kappa = 0.80 pANCA Percent agreement (95% confidence)


QUANTA Flash®MPO CIA

Positive 30 5* 35 Pos agreement 93.8% (79.2-99.2%)

Negative 2# 34 36 Neg agreement 87.2% (72.6-95.7%)


*3 samples from MPA and 2 from GPA patients #1 sample from MPA and 1 from GPA patients

Table 2. Agreement between QUANTA Flash and IIF

Comparison of QUANTA Flash CIA with IIF

The results of the QUANTA Flash PR3 and MPO CIA werecompared to IIF results. MPO-ANCA was analyzed vs.the presence of a pANCA pattern and PR3-ANCA vs. thepresence of cANCA. In the entire cohort, 28 samplesshowed a cANCA pattern in IIF, and 34 were positiveby CIA.14 For MPO-ANCA, 32 samples showed a pANCApattern, and 35 were positive by CIA. The agreement withMPO-ANCA was 90.1% for CIA (Table 2).14 For PR3-ANCAthe agreement was 91.5% for CIA (Table 2).14

Comparison of QUANTA Lite ELISA and

QUANTA Flash CIASeventy one samples (41 GPA and 30 MPA, N=71) wereused to compare the PR3 and MPO ANCA QUANTAFlash CIA with QUANTA Lite® PR3 and MPO ELISA.14 The positive agreement of the PR3 CIA compared toELISA was 100%, the negative agreement 92.5%, andthe overall agreement 95.8% (Table 1).14 The positivepercent agreement of the MPO CIA compared to theELISA was 100%, the negative agreement 85.7%, andthe overall agreement 91.5% (Table 1).14


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Reference List

1. Wegener F:On generalised septic vessel diseases. By Friedrich Wegener,1937 Thorax 1987;42:918-919.

2. Falk RJ, Gross WL, Guillevin L, Hoffman GS, Jayne DR, Jennette JC et al.:Granulomatosis with polyangiitis (Wegener’s): an alternative name forWegener’s granulomatosis. Arthritis Rheum 2011, 63: 863-864.

3. Davies DJ, Moran JE, Niall JF, Ryan GB: Segmental necrotisingglomerulonephritis with antineutrophil antibody: possible arbovirusaetiology? Br Med J (Clin Res Ed) 1982, 285: 606.

4. van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van EsLA et al.: Autoantibodies against neutrophils and monocytes: tool fordiagnosis and marker of disease activity in Wegener’s granulomatosis.Lancet 1985, 1: 425-429.

5. Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies withspecificity for myeloperoxidase in patients with systemic vasculitis andidiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med1988, 318: 1651-1657.

6. Niles JL, McCluskey RT, Ahmad MF, Arnaout MA: Wegener’sgranulomatosis autoantigen is a novel neutrophil serine proteinase.Blood 1989, 74: 1888-1893.

Diagnostic methods for ANCA: past and present...Continued

7. Conrad K, Schößler W, Hiepe F, Fritzler M: Myeloperoxidase Antibodies.In Autoantibodies in Systemic Autoimmune Diseases- A DiagnosticReference. 2 edition. Edited by Conrad K, Schößler W, Hiepe F, Fritzler MPabst; 2007:111-113.

8. Conrad K, Schößler W, Hiepe F, Fritzler M: Proteinase 3 Antibodies.In Autoantibodies in Systemic Autoimmune Diseases- A DiagnosticReference. 2 edition. Edited by Conrad K, Schößler W, Hiepe F, Fritzler MPabst; 2007:147-149.

9. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ et al.:International Consensus Statement on Testing and Reporting ofAntineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999,111: 507-513.

10. Wang G, Csernok E, De GK, Gross WL: Comparison of eight commercialkits for quantitation of antineutrophil cytoplasmic antibodies (ANCA). JImmunol Methods 1997, 208: 203-211.

11. Csernok E, Ahlquist D, Ullrich S, Gross WL: A critical evaluation ofcommercial immunoassays for antineutrophil cytoplasmic antibodiesdirected against proteinase 3 and myeloperoxidase in Wegener’sgranulomatosis and microscopic polyangiitis. Rheumatology (Oxford)2002, 41: 1313-1317.

12. Schulte-Pelkum J, Offermann N, Fooke M: New highly sensitiveand specific Lateral Flow Tests for the detection of Proteinase 3,Myeloperoxidase and Glomerular Basement Membrane antibodies. 8thInternational Congress on Autoimmunity May 9-13th 2012, Granada,Spain 2012, Poster: 234.

13. Lucassen R, Schulte-Pelkum J, Petschinka M, Fooke M: New sensitiveand reliable Lateral flow assay for the detection of proteinase 3 andmyeloperoxidase antibodies. Report on the 10th Dresden Symposiumon Autoantibodies 2011, Poster: 694-695.

14. Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L et al.:Development and performance evaluation of novel chemiluminescenceassays for detection of anti-PR3 and anti-MPO antibodies. Clin ChimActa 2012, 413: 719-726.

15. Csernok E, Holle J, Hellmich B, Willem J, Tervaert C, Kallenberg CG et al.:Evaluation of capture ELISA for detection of antineutrophil cytoplasmicantibodies directed against proteinase 3 in Wegener’s granulomatosis:first results from a multicentre study. Rheumatology (Oxford) 2004, 43:174-180.

16. Avidin-Biotin Chemistry: A Handbook. 2008.17. Roggenbuck D, Buettner T, Hoffmann L, Schmechta H, Reinhold

D, Conrad K: High-sensitivity detection of autoantibodiesagainst proteinase-3 by a novel third-generation enzyme-linkedimmunosorbent assay. Ann N Y Acad Sci 2009, 1173: 41-46.

18. Damoiseaux J, Dahnrich C, Rosemann A, Probst C, Komorowski L,Stegeman CA et al.: A novel enzyme-linked immunosorbent assay usinga mixture of human native and recombinant proteinase-3 significantlyimproves the diagnostic potential for antineutrophil cytoplasmicantibody-associated vasculitis. Ann Rheum Dis 2009, 68: 228-233.

19. Radice A, Bianchi L, Gliona S, Trezzi B, Maggiore U, Sinico RA:Comparison of PR3 specific ANCA Assays performance for diagnosis ofGranulomatosis with polyangiitis (GPA). 16th International Vasculitisand ANCA Workshop 2013, -Poster.

20. Posthuma-Trumpie GA, Korf J, van AA: Lateral flow (immuno)assay: itsstrengths, weaknesses, opportunities and threats. A literature survey.Anal Bioanal Chem 2009, 393: 569-582.

21. Bonenberger J, Doumanas M: Overcoming sensitivity limitationsof lateral-flow immunoassays with a novel labeling technique. IVD

Technology 2006.22. Gordon J, Michel G: Analytical sensitivity limits for lateral flow

immunoassays. Clin Chem 2008, 54: 1250-1251.23. Damoiseaux J, Vaessen M, Knapen Y, Csernok E, Stegeman CA, Van

PP et al.: Evaluation of the FIDIS vasculitis multiplex immunoassayfor diagnosis and follow-up of ANCA-associated vasculitis andGoodpasture’s disease. Ann N Y Acad Sci 2007, 1109: 454-463.



• Many algorithms have been proposed asthe best possible way to detect ANCA. TheIIF screening on fixed neutrophils followedby verification of positive results remainsthe recommended method according to theInternational Consensus Statement on ANCA Testing and Reporting.

• ELISA methods often are burdened with batch-ing and relatively long result times. Accurate,rapid diagnosis followed by appropriate

therapy is paramount to halting the deleteriouseffects of ANCA vasculitic disease. Many clini-cians now desire assays which allow quick turn-around time along with diagnostic accuracy.

• QUANTA Flash PR3 and MPO CIAs are sensi-tive and specific assays for rapid detection (30min) of PR3 and MPO ANCA, which fulfills thecompelling need for a rapid detection system.Moreover, these CIAs allow quantitative detec-tion of antibodies with a broad assay range.



Introduction

Antineutrophil cytoplasmic antibodies (ANCA) area heterogeneous group of autoantibodies whoseantigenic targets include proteinase-3 (PR3) and myelo-peroxydase (MPO).1 Detection of these autoantibodies

is useful for the diagnosis and follow-up of small vesselvasculitis such as granulomatosis with polyangiitis(GPA), microscopic polyangiitis (MPA), and eosinophilicgranulomatosis with polyangiitis (EGPA).2 Theseautoantibodies and their respective prevalence arereported in Table 1.3

PR3 and MPO autoantibodies can be detected usingvarious techniques and different multi step strate-gies.4 The first step is typically a screening test usingan indirect immunofluorescence (IIF) assay on humanneutrophils which have been fixed with ethanol.

Autoantibodies against antigens other than PR3 andMPO can cause positive results with ANCA testing byIIF and the sensitivity of IIF is not perfect. Thereforeconfirmation and identification of PR3 and MPO ANCAantibodies is very important and can be obtained usingdifferent solid phase technologies such as immunodot,

enzyme linked immunosorbent assay (ELISA), multiplexassays, or more recently by chemiluminescent immuno-assay (CIA). Just as these autoantibodies are usefultools for the diagnosis of small vessel vasculitis, theyare also useful markers for monitoring disease activ-ity and therapy efficacy.5,6 Providing numerical labora-tory results across a broad detection range is especial-ly helpful for the management of these diseases.Furthermore, when patients present with critical acutesymptoms having quantitative results can further aid inmaking a differential diagnosis between other condi-tions including infection.

Limitations of IIF and ELISA for certainpatient subsets

Even though IIF assays are sensitive and widelyaccepted as the first step method to be used in routinepractice for ANCA screening, they are time consumingfor technicians and inappropriate in emergency settings.Moreover, some discrepancies between IIF and specif-ic tests for identification of the target of these antibod-ies have been reported, mainly in patients undergoingimmunosuppressive treatment. Like any IIF assay, wideintra- and inter-laboratory variability is also observed inANCA testing. This is due to variability from a numberof factors including; sources of neutrophils, neutro-phil preparation and fixation, the conjugate reagent,the reading system used and finally the subjectivityand the expertise of the technician. Different PR3 andMPO preparations and various methodologies are usedto detect these antibodies that specifically recognizetargets with conserved conformational epitopes andovercome the challenges of IIF for patient monitoring.7, 8

Lucile Musset, PhD

Director Laboratory of Immunochemistry

Pitie Salpetriere Hospital

Paris, France

Makoto Miyara, MD, PhD

Sr. Lecturer

Pitie Salpetriere Hospital

Paris, France

ANCA testingin emergencysetting

PR3 (%) MPO (%)

Granulomatosis withpolyangiitis

66 24

Microscopic polyangiitis 26 58

Eosinophilic granulamtosiswith polyangiitis

10 60

Table 1. Prevalence of PR3 and MPO-ANCA in different groups ofsmall vessel vasculitis.


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ANCA testing in emergency setting...Continued


ANCA testing in an emergency setting

ANCA associated vasculitidies are chronic, multi-system-ic, disorders often affecting several organs, character-

ized by flares and remissions. However, despite thischronic nature, ANCA testing can be requested inemergency cases during acute stage flare-ups. Suchtesting is critical in order to quickly support a differ-ential diagnosis and initiate appropriate immunosup-pressive therapies in order to avoid irreversible organdamage.4, 9 Additionally, the clinical manifestations ofvasculitis can be severe including alveolar hemorrhage,glomerulonephritis, scleritis, and necrotizing sinusitis. Therefore, it is of the utmost importance to report ANCAresults in a timely manner to aid in prompt diagnosisand treatment.

Since relapse of ANCA associated vasculitis occursin 30-60% of cases, ANCA testing is also required forfollow-up. According to disease stage and activity, serialtesting is required at intervals of several months (threeto six months). Furthermore, ANCA testing is usuallyordered to assess the side effects of immunosuppres-sive therapy, such as malignancies, or for differentialdiagnosis with undercurrent infectious diseases.10,11

Rapid and accurate results using theBIO-FLASH® chemiluminescent analyzer

We have recently analyzed a chemiluminescence immuno-

assay developed on an automated analyzer for thedetection of PR3 and MPO-ANCA. A retrospective studyfrom 74 sera corresponding to 45 patients with ANCAassociated vasculitis (32 GPA, 13 MPA) and 62 sera fromhealthy and control diseases (systemic lupus erythemato-sus, Sjögren’s syndrome, healthy donors) was performed.Sera were tested using the QUANTA Flash® PR3 and MPOassays on the BIO-FLASH® chemiluminescent analyz-er (INOVA Diagnostics). Results were compared to thoseobtained using routine multiplex assays Fidis TM VasculitisPanel (BMD) performed using the Luminex analyser in ourlaboratory (Figure 1).

The sensitivity and the specificity of the QUANTA Flashassay was 78.4% and 83.4%, respectively. The sensitiv-ity and the specificity of routine technique was 73% and80.1%, respectively. A good agreement was observedfor MPO (92.9% kappa =0.82) and for PR3 (96.4%kappa=0.92). Four samples were PR3-ANCA positive byQUANTA Flash and negative by multiplex. Eight sampleswere MPO-ANCA positive by multiplex and negative

Figure 1. Comparison of PR3 and MPO-ANCA tested on BIO-FLASH and Fidis Vasculitis Panel.Units are expressed as Calculated Units (CU) and Luminex Units (LU/mL)



by QUANTA Flash (Figure 2). Those samples were fromfour patients with treated ANCA associated vasculitis(two GPA, one mononeuritis simplex and one alveo-lar haemorrhage) and four patients with inflamma-

tory/autoimmune diseases (Grave’s disease, psoriaticrheumatism, giant cell arteritis and Sjögren’s syndrome).Four samples were PR3-ANCA positive by CIA andnegative by multiplex, coming from patients withtreated ANCA-associated vasculitis (two GPA, one MPAand one EGPA).

Multiplex

PR3 Neg Pos Total

B I O - F L A S H

Neg 70 0 70

Pos 4 39 43

Total 74 39 113

Multiplex

MPO Neg Pos Total

B I O - F L A S H

Neg 79 8 87

Pos 0 26 26

Total 79 34 113

Figure 2. Concordance analysis for PR3 and MPO-ANCA tested onBIO-FLASH and Fidis Vasculitis Panel. Neg: negative; Pos: positive.

Utility of BIO-FLASH in an emergencysetting

For assessment of practicability of the BIO-FLASH in

an emergency setting, inexperienced residents at ourhospital were recruited to evaluate the functional-ity and ease of use of BIO-FLASH. After a short brief-ing lasting 15 minutes, the residents were able toperform the analysis and retrieve results within 30minutes. The QUANTA Flash PR3 and MPO assays on theBIO-FLASH system display good sensitivity and specific-ity combined with high agreement to routine methods.Moreover, the retrieval of results was rapid and easy toobtain, making the technique suitable for emergen-cy settings as well as for the follow-up of patients inroutine practice.


• ANCA testing by IIF is an important first stepwhen screening, positive results or cases ofstrong clinical suspicion should be run on asolid phase assay for confirmation.

• When monitoring disease activity or therapyefficacy, a methodology other than IIF shouldbe used to allow for numerical results.

• Acute disease flares can be confounding and

adequate testing methods in an emergencysetting can impact patient outcomes.

• A method such as BIO-FLASH which is rapid,accurate, and can be performed by all levels oflaboratory staff is ideally suited for urgent aswell as monitoring requests for ANCA testing.

References

1. Kallenberg CG. Pathogenesis of ANCA-associated vasculitis. Ann RheumDis 2011 ; 70 Suppl1 : i59-63.

2. Watts RA, Scott DG. Recent developments in the classification and

assessment of vasculitis. Best Pract Res Clin Rheumatol. 2009;23:429-43.3. Hagen EC, Daha MR, Hermans J, Andrassy K, Csernok E, Gaskin G et al.Diagnostic value of standardized asays for anti-neutrophil cytoplasmicantibodies in idiopathic systemic vasculitis. Kidney Int 1998; 53:743-53.

4. Vermeersch P, Vervaeke S, Blockmans D, et al. Determination ofanti-neutrophil cytoplamsic antibodies in small vessel vasculitis:comparative analysis of different strategies. Clin Chim Acta 2008;397:7781.

5. Hellmich B, Flossmann O, Gross WL et al. EULAR recommendations forconducting clinical studies and/or clinical trials in systemic vasculitis:focus on anti-neutrophil cytoplasm antibody-associated vasculitis. AnnRheum Dis 2007; 66: 605-17.

6. Mukhtyar C, Guillevin L, Cid MC, Dasgupta B, De Groot K, Gross WL etal. EULAR recommendations for the management of primary small andmedium vessel vasulitis. Ann Rheum Dis 2009; 68: 310-7.

7. Damoiseaux J, Dahnrich C, Rosemann A et al. A novel enzyme-linked immunosorbent assay using a mixture of human native and

recombinant protéinase-3significantly improves the diagnosticpotential for antineutrophil cytoplasmic antibody-associated vasculitis.Ann Rheum Dis 2009;68:228-33.

8. Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L, Sinico RA.Development and performance evaluation of novel chemiluminescenceassays for detection of anti-PR3 anti-MPO antibodies. Clin Chim Acta2012; 413: 719-26.

9. Seck SM, Dussol B, Brunet P, Burtey S. Clinical features and outcomesof ANCA-associated renal vasculitis. Saudi J Kidney Dis Transpl. 2012Mar;23(2):301-5.

10. Terrier B, Saadoun D, Sène D, Ghillani P, Amoura Z, Deray G, Fautrel B,Piette JC, Cacoub P. Antimyeloperoxidase antibodies are a useful markeof disease activity in antineutrophil cytoplasmic antibody-associatedvasculitides. Ann Rheum Dis 2009; 68:1564-1571.

11. Luxton G, Langham R. ANCA serology in the diagnosis and managmentof ANCA-associated renal vasculitis. Nephrology. 2008;13 (Suppl2):S17-S23.



A new look atANCA testingwith NOVAView®*Gabriella Lakos, MD, PhD

Medical Director INOVA Diagnostics

San Diego, CA

Carol Buchner, MT (ASCP)

Manager, IFA Development

INOVA Diagnostics

San Diego, CA

Introduction

The International Consensus Statement on Testing andReporting of Antineutrophil Cytoplasmic Antibodies(ANCA)1, 2 states that the minimum requirements forANCA testing is that “IIF should be performed on all serafrom new patients, since 10% of ANCA positive serain patients with Wegener’s granulomatosis or micro-scopic polyangiitis can be demonstrated only by IIF”.(Wegener’s granulomatosis has recently been renamedas granulomatosis with polyangiitis (GPA)3). In spiteof these recommendations, laboratory strategy for thedetection of ANCA varies across laboratories accord-

ing to geographical areas, traditions and local experi-ence. New antigen-specific solid phase assays are highlysensitive and specific,4 but the performance characteris-tics of available assays vary widely.5, 6

Challenges of IIF method for detectionof ANCA

The IIF method (whether it is used for the detection of

antinuclear antibodies (ANA), ANCA, or organ-specif-ic antibodies) occupies a special – not very popular –place in the laboratory. First, both slide preparation andslide reading requires significant manual processes,which is time consuming and prone to technical issues.

The manual nature of slide preparation and slidereading, coupled with handwritten transcription ofresults in the dark room is the primary source of samplemixups and erroneous result reporting. Furthermore,some (predominantly homogeneous) ANA samplesmay produce patterns identical to those produced by

pANCA. This is one of the reasons why the interpreta-tion of ANCA IIF images is challenging and requires thereview of results on both ethanol and formalin fixedsubstrates.

After successful launch of the ANA module on NOVAView®, INOVA Diagnostics has developed a new appli-cation that allows the use of NOVA View automatedfluorescent microscope for ANCA testing. NOVA Viewwith ANCA software module and NOVA Lite ANCAreagents are used together as a system to bring ANCAIIF testing to a level that meets the expectations of

modern autoimmune laboratories.

NOVA View® with ANCA module

The NOVA View® system contains a fluorescent micro-scope with a motorized stage, LED light source, and aCCD (charge-coupled device) digital camera. The NOVAView software controls the microscope and the camera. The system acquires high resolution digital images ofthe substrate, archives them, and displays them on thecomputer screen for the user for review. NOVA Viewmeasures the light intensity of the images, and prelimi-

narily categorizes the samples as negative or positiveaccording to a pre-programmed cutoff. The NOVA Viewsoftware also provides pattern recognition for positivesamples based on software algorithm. The ability ofthe operator to manage (enlarge, overlay) the acquireddigital images facilitates interpretation. Importantly,NOVA View does not report final results withoutconfirmation from a trained technician.

The NOVA Lite® Ethanol ANCA kit with DAPI and NOVALite Formalin ANCA kit with DAPI are enhanced versionsof the NOVA Lite ANCA kits, that are IVD licensed for

*NOVA View is not available for sale in the US



manual use. Cell density in the wells was increased andstandardized. The conjugate in the kit contains DAPI(4’,6-diamidino-2-phenylindole), a blue fluorescent dyethat stains the nuclei. As images are taken by NOVA

View with both the DAPI and the FITC filters, everyimage is available in both “colors” which can be overlaidto facilitate pattern identification, and can be used toconfirm the presence of cells on negative wells.

Each kit contains twenty, 12-well slides (Figure 1). Eachslide contains a two-dimensional barcode that desig-nates the slide type (ethanol- or formalin-fixed, 12-well),and has a unique identifier, allowing the identifica-tion of the slide and linking of patient informationto the wells (Figure 1). These slides can be processedby automated slide processing instruments, such as

QUANTA-Lyser®. QUANTA-Lyser is programmed to readIFA slide barcodes to confirm the substrate type, andto match patient information received from thelaboratory information system (LIS) to a specific welllocation on the slide.

These unique features ensure sample traceability, anddeliver positive patient identification for IFA process-ing and analysis. QUANTA Link®, a middleware softwarefacilitates communication between the LIS, QUANTA-Lyser, and other lab instruments, thereby providing thefoundation of a fully integrated autoimmune laboratory.

ANCA module characteristics and userinterface

During slide reading, NOVA View analyzes at least 25 cells,and acquires at least three representative digital images.Very bright samples will undergo a second imaging,when images are captured with reduced (optimal)exposure time to facilitate pattern recognition. Afterfinishing the imaging process, the measured LIU andthe suggested result (negative/positive and pattern) are

displayed for the user. NOVA View identifies nuclear andcytoplasmic staining patterns. In the case of non-charac-teristic or mixed patterns (such as in the presence ofcertain ANAs), NOVA View reports positive result with

unrecognized patterns.

Use of the NOVA Lite ANCA DAPI kits with QUANTA-Lyser and NOVA View simplifies andstreamlines the IIF reading/interpretation workflow, by:

• Reducing hands-on time in the slide reading process.

• Supporting the interpretation of the samples withobjective information.

• Eliminating the need for darkroom.

• Eliminating transcription errors withbarcoded slides.

• Separating image acquisition from imageinterpretation.

Figure 1. 12-well barcoded ANCA slides containingethanol-fixed (E-ANCA) and formalin-fixed(F-ANCA) granulocytes.



A new look at ANCA testing... Continued

Figure 2 shows a representative screen shot of a pANCApositive sample, as the user sees it. During the imagereview process, the operator can enlarge and overlayDAPI and FITC images (Figure 2). After review, the opera-

tor has two choices: either confirming the NOVA Viewsuggested result, or revising it (regarding both negativ-ity/positivity and pattern) and then confirming. Thesoftware contains five customizable buttons that theusers can pre-program according to their local report-ing nomenclature (for example, pANCA, cANCA, atypi-cal ANCA, Possible ANA, or See Comment). For eachsample, a comment can be entered that becomes partof the reported results. Results can be transferred to theLIS system or a patient report can be directly generatedby the NOVA View.

QUANTA Link and the Multi-Analyte Screen

QUANTA Link can greatly enhance the IIF experi-ence with the NOVA View, and facilitates ANCA inter-

pretation. One important feature of QUANTA Link isthe Multi-Analyte Screen. This feature brings togeth-er digital images generated on the same sample byvarious assays. In the case of ANCA, images obtainedon ethanol-fixed and formalin-fixed substrates can bedisplayed next to each other, and if HEp-2 ANA resultsare available, this can also be added to the same screen.Having these images on the same screen at the sametime allows the interpretation of ANCA IIF results withhigh accuracy. Figure 3 shows an example that wasgenerated on a cANCA and speckled ANA doublepositive sera sample.

Performance characteristics

During the establishment and validation of the ANCAmodule performance, the cut-off LIU was determinedseparately for ethanol- and formalin-fixed slides on 120reference specimens.

• Precision for both substrate types was examinedon three samples, run for five days in triplicate withtwo runs per day. All results were within one gradedifference based on digital image reading.

• Accuracy was assessed by determining theendpoint titer of six positive samples based on LIUvalue, digital image reading, and manual imagereading. Endpoints were within one dilution stepfrom each other in each comparison.

• In a clinical validation study, samples from 80healthy controls, 20 infectious disease patients,and 21 MPO/PR3 positive patients were assayed.Performance is shown in Table 1.

Figure 2. Representative user interface showing a DAPI/FITCoverlaid image of a pANCA positive sample.

Figure 3. Representative screenshot of the Multi-AnalyteScreen from QUANTA Link showing a cANCA andspeckled ANA double positive sample.



% sensitivity or specificity (number of positive/tested samples)

Characteristics Formalin-fixed neutrophil substrate. Ethanol-fixed neutrophil substrate.

Relative sensitivity based on LIU cutoff 76.2% (16/21) 90.5% (19/21)

Relative sensitivity based on digital image reading 90.5% (19/21) 95.2% (20/21)

Relative specificity based on LIU cutoff 100.0% (80/80) 96.3% (77/80)

Relative specificity based on digital image reading 100.0% (80/80) 90.0% (72/80)

Agreement between LIU-based NOVA View resultsand digital image reading

96.7% (117/121) 93.3% (112/120)

Agreement between digital image reading andmanual (microscopic) reading

90.9% (110/121) 93.4% (113/121)

References

1. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, Jayne DJennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, vander Woude F, Wieslander J, Wiik A. International Consensus Statement on

Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA).Am J Clin Pathol. 1999;111:507-13.

2. Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, McEvoyR, Pusey C, Pollock W, Trevisin M, Wiik A, Wong R; International Group forConsensus Statement on Testing and Reporting of Antineutrophil Cyto-plasmic Antibodies (ANCA). Addendum to the International ConsensusStatement on testing and reporting of antineutrophil cytoplasmic anti-bodies. Quality control guidelines, comments, and recommendations fortesting in other autoimmune diseases. Am J Clin Pathol. 2003;120:312-8.

3. Falk RJ, Gross WL, Guillevin L, Hoffman GS, Jayne DR, Jennette JC, Kal-lenberg CG, Luqmani R, Mahr AD, Matteson EL, Merkel PA, Specks U, WattRA; American College of Rheumatology; American Society of NephrologyEuropean League Against Rheumatism. Granulomatosis with polyangiitis(Wegener’s): an alternative name for Wegener’s granulomatosis. Arthritis

Rheum. 2011;63:863-4.4. Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L, Sinico RA.

Development and performance evaluation of novel chemiluminescenceassays for detection of anti-PR3 and anti-MPO antibodies. Clin Chim Acta2012;413:719-26.

5. Wang G, Csernok E, de Groot K, Gross WL. Comparison of eight commer-cial kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA)J Immunol Methods. 1997;208:203-11.

6. Holle JU, Herrmann K, Gross WL, Csernok E. Comparative analysis ofdifferent commercial ELISA systems for the detection of anti-neutrophilcytoplasm antibodies in ANCA-associated vasculitides. Clin Exp Rheuma-tol. 2012;30(1 Suppl 70):S66-9.

IN CONCLUSIONIn conclusion :• NOVA View is an automated solution for

autoimmune laboratories performing IIF assays.It simplifies and streamlines the IIF reading/interpretation workflow, and increases thesafety of IIF testing by sample traceability.

• The newly developed ANCA module acquireshigh resolution digital images on ANCA slides,

and provides the user with tools that facilitiesimage interpretation.

• The ANCA module showed reliable and repro-ducible performance during evaluation studies.

Table 1. Clinical performance of NOVA View with formalin-fixed and ethanol-fixed neutrophil substrate


16/20

Anti-PR3antibodiesin ulcerativecolitis


Michael Mahler, PhDVice President Research & Development

INOVA Diagnostics, San Diego, CA

Gary L. Norman, PhD

Director Research & Development

INOVA Diagnostics, San Diego, CA

Marcos Lopez-Hoyos, MD, PhD

Director Clinical Laboratory Hospital Universitario

Marques de Valdecilla

Santander, Spain

Introduction

Antineutrophil cytoplasmic antibodies (ANCA) direct-ed against proteinase 3 (PR3) or myeloperoxidase(MPO) are used in the diagnostic workup of small vesselvasculitis (SVV), such as granulomatosis with polyangi-itis (GPA), and microscopic polyangiitis.1 Conventionalscreening for anti-PR3- and MPO-antibodies startswith the indirect immunofluorescence assay (IIF) onethanol-fixed human neutrophils followed by confirma-

tory ELISA.1, 2 Anti-PR3 antibodies generate a cytoplas-mic staining pattern (cANCA) on ethanol-fixed humanneutrophils, whereas anti-MPO antibodies generate aperinuclear staining (pANCA) pattern.1 Many laborato-ries also evaluate specimens on formalin-fixed humanneutrophils.3

Biomarkers for inflammatory bowel disease

An atypical pANCA pattern, sometimes referred to asxANCA,4 is found in patients with inflammatory bowel

disease (IBD), mainly in patients with ulcerative colitis(UC). When combined with anti-Saccharomyces cerevisi-ae antibodies (ASCA), atypical pANCA has been recom-mended as a way to help distinguish UC from Crohn’sdisease (CrD).3, 5-9 ASCA sero-positivity is a predominantfeature of CrD, while atypical pANCA is a marker of UC.3,7

Both ASCA and ANCA have been reported to predictthe development of IBD.10 Despite many studies, thespecificity of ANCA in IBD remains poorly defined.5, 11 The diagnosis of IBD including UC and CrD is largelybased on endoscopic and histological assessment of theinflamed tissue.5, 12 While several antibody tests can assist

in the diagnosis of CrD including (ASCA) and pancreaticmajor zymogen granule protein 2 (MZGP2),13-15 the onlyserological biomarker for UC is atypical pANCA detect-ed by IIF.3 However, IIF is time consuming and observ-er-dependent, has low throughput requiring highly-trained personnel, can generate significant variation16 and demonstrates limited specificity.6, 17 IIF is also unableto provide information about ANCA antigen specificity.5,6 Several studies have aimed to identify the major targetantigen of atypical pANCA in IBD,11, 18 but major disease-specific target antigens are are yet to be identified.

Detection methods for PR3-ANCA

Over the last decade, a variety of different method-ologies have been developed and commercialized forthe detection of anti-PR3 antibodies, including IIF onhuman neutrophils, ELISA,19 line immunoassays (LIA),20 capture21-22 and anchor ELISAs23-24 as well as multiplexassays25-27 using native purified PR3 and, more recently,recombinant PR3 antigen.28 Despite several compara-tive studies, it remains debatable as to which method-ology for anti-PR3 antibodies detection provides the

Marker Ulcerative colitis Crohn’s disease

pANCA 40-60% 5-20%

ASCA IgG 5-10% 40-70%

ASCA IgA 5-10% 40-70%

PR3-ANCA 15-40% 0-10%

MZGP2 0-5% 20-30%

Table 1. Prevalence of autoantibodies in ulcerative colitis andCrohn’s disease5, 14



highest clinical accuracy for the diagnosis of GPA.19, 29 Several studies published over the last decade suggest-ed that the sensitivity of both capture as well as novelanchor assays were superior to classical ELISA and evento IIF.19, 23-24, 30-31

PR3-ANCA as a marker for UC

In contrast to historical data,32 more recent studiesreported anti-PR3 antibodies in a significant percent-age of IBD patients.27 This raises the possibility thatakin to anti-PR3 antibodies as a marker for SVV, anti-PR3 antibodies may also be a marker for IBD. Recentlyit was suggested that anti-PR3 antibodies measuredby a novel chemiluminescent immunoassay (CIA) on a

random access auto-analyzer (BIO-FLASH®) are usefulin the differential diagnosis of UC and CrD.33 Our obser-vation that anti-PR3 antibodies can be detected insera from patients with IBD, with higher prevalence inUC vs. CrD patients, suggests that anti-PR3 antibodiestesting could assist in discriminating UC from CrD, andin discriminating IBD from other gastrointestinal condi-tions. The terms “indeterminate colitis” or “IBD unclassi-fied” (IBD-U) categorize patients in whom the diagnosisof UC or CrD is not clear.5, 17, 34 The differential diagno-sis may be complicated in patients with irritable bowel

• Whole GI tract (terminal ileum)

• Discontinuous

• Transmural inammation

• Extraintestinal manifestations (skin,

eyes, joints) and colorectal malignancy

• Only large bowel

• Continuous

• Mucosal inammation

• Extraintestinal manifestations (skin,

eyes, joints) and colorectal malignancy

Crohn’s

Disease (CrD)

Ulcerative

Colitis (UC)

pANCA

PR3

ASCA IgG/IgA

MZGP2

IndeterminateColitis

UC-like

CD

E

F

C

Figure 1. Differentiation between ulcerative colitis (UC) and Crohn`s disease (CrD. The clinical difference between UC and CrD and thepotential role of PR3-ANCA as a marker to help in the diagnosis of UC is illustrated

syndrome, celiac disease, or other colorectal diseases, withfeatures indistinguishable from those seen in patientswith IBD.5

Studies of anti-PR3 antibodies in IBD are limited andhave been based on relatively small cohorts of UCpatients.27, 35-36 When twelve anti-PR3 antibody assayswere compared using 22 IBD sera, the reported preva-lence of anti-PR3 antibodies ranged from 4% to 43%,raising concerns as to the reliability of the assays used inthese studies.27, 35-36 The two assays with highest sensi-tivity (BINDAZYME Anti-PR3 39% and Rainbow ELISAPR3 43%) also showed the lowest specificity (88%). In arecent study it was found that anti-PR3 antibodies arefound in patients with IBD, albeit at lower titers than inpatients with GPA. This was demonstrated using threeseparate and independent fully automated platformsfor autoantibody detection. The prevalence of theantibodies differed between the methods used, butthis was mainly related to the way the manufacturer-specific cut-off was defined (focused on high specificityfor GPA).



Anti-PR3 antibodies in ulcerative colitis...Continued

Differentiation between PR3-ANCA positiveUC and GPA patients

The fact that anti-PR3 antibodies are found in UC could

be interpreted as compromising the specificity of anti-PR3 antibodies for GPA. Anti-PR3 antibodies in patientswith GPA are often associated with a cANCA pattern onethanol-fixed neutrophils, while in UC patients an atypi-cal pANCA is most often observed. The latter is mostlikely explained by reactivity to other antigens that havebeen reported in the past to be associated with UC.37

Interestingly, recent studies have described patientswith overlapping features of UC and GPA.38-40 To whatextent anti-PR3 antibody positive patients with UC willdevelop full-blown SVV over the course of their disease,

needs to be assessed in large longitudinal studies. It iswidely appreciated that different autoimmune diseas-es can overlap in some patients, which was recentlydescribed as poly-autoimmunity.41 Therefore, addition-al studies are required to determine whether there is anoverlap between the two chronic inflammatory diseas-es. While GPA typically affects the upper respiratorytract and the kidneys, UC typically is an inflammatorydisease limited to the colon. Although 10% of patientswith SVV can present with ulcerations of the colon,42 isolated gastro intestinal tract involvement is infre-quently seen in ANCA-positive patients with SVV.43

Reference List

1. Wiik A: What you should know about PR3-ANCA. An introduction.Arthritis Res 2000, 2: 252-254.

2. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, JayneD, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, SilvestriniR, van der Woude F, Wieslander J, Wiik A: International ConsensusStatement on Testing and Reporting of Antineutrophil CytoplasmicAntibodies (ANCA). Am J Clin Pathol 1999, 111: 507-513.

3. Lochman I, Kral V, Lochmanova A, Lupac J, Cebecauer L: ANCA in thediagnosis of neutrophil-mediated inflammation. Autoimmun Rev 2011,10: 295-298.

4. Papp M, Altorjay I, Lakos G, Tumpek J, Sipka S, Dinya T, Palatka K, VeresG, Udvardy M, Lakatos PL: Evaluation of the combined applicationof ethanol-fixed and formaldehyde-fixed neutrophil substrates foridentifying atypical perinuclear antineutrophil cytoplasmic antibodiesin inflammatory bowel disease. Clin Vaccine Immunol 2009, 16: 464-470

5. Papp M, Norman GL, Altorjay I, Lakatos PL: Utility of serologicalmarkers in inflammatory bowel diseases: gadget or magic? World JGastroenterol 2007, 13: 2028-2036.

6. Bossuyt X: Serologic markers in inflammatory bowel disease. Clin Chem2006, 52: 171-181.

7. Reese GE, Constantinides VA, Simillis C, Darzi AW, Orchard TR, FazioVW, Tekkis PP: Diagnostic precision of anti-Saccharomyces cerevisiaeantibodies and perinuclear antineutrophil cytoplasmic antibodies ininflammatory bowel disease. Am J Gastroenterol 2006, 101: 2410-2422.

8. Quinton JF, Sendid B, Reumaux D, Duthilleul P, Cortot A, GrandbastienB, Charrier G, Targan SR, Colombel JF, Poulain D: Anti-Saccharomycescerevisiae mannan antibodies combined with antineutrophilcytoplasmic autoantibodies in inflammatory bowel disease: prevalenceand diagnostic role. Gut 1998, 42: 788-791.

9. Ferrante M, Henckaerts L, Joossens M, Pierik M, Joossens S, Dotan N,Norman GL, Altstock RT, Van SK, Ru tgeerts P, Van AG, Vermeire S: Newserological markers in inflammatory bowel disease are associated withcomplicated disease behaviour. Gut 2007, 56: 1394-1403.

10. Israeli E, Grotto I, Gilburd B, Balicer RD, Goldin E, Wiik A, ShoenfeldY: Anti-Saccharomyces cerevisiae and antineutrophil cytoplasmicantibodies as predictors of inflammatory bowel disease. Gut 2005, 54:1232-1236.

11. Terjung B, Spengler U, Sauerbruch T, Worman HJ: “Atypical p-ANCA”in IBD and hepatobiliary disorders react with a 50-kilodaltonnuclear envelope protein of neutrophils and myeloid cell lines.Gastroenterology 2000, 119: 310-322.


• The only serological marker for UC is atypicalpANCA detected by IIF. However, IIF is observer-dependent and is unable to provide informationabout ANCA antigen specificity.

• Anti-PR3 antibodies are present in a significantpercentage of IBD patients and may be a markerof concurrent SVV-related disease.

• Anti-PR3 antibodies measured by BIO-FLASH

CIA are useful in the differential diagnosis ofUC and CrD.

Abbreviations

ASCA, anti-Saccharomyces cerevisiae antibody; AUC, Area under thecurve; cANCA, cytoplasmic anti-neutrophil cytoplasmic antibodies;CIA, chemiluminescence assay; CrD, Crohn`s disease; CU, calculat-ed units; GPA, granulomatosis with polyangiitis; IBD, inflammato-ry bowel disease; IIF, indirect immunofluorescence assay; LIA, lineimmunoassay; LR, likelihood ratio; pANCA, perinuclear anti-neutro-

phil cytoplasmic antibodies ROC, Receiver Operating Characteristic;UC, ulcerative colitis; WG, Wegener’s granulomatosis



12. Mowat C, Cole A, Windsor A, Ahmad T, Arnott I, Driscoll R, Mitton S,Orchard T, Rutter M, Younge L, Lees C, Ho GT, Satsangi J, Bloom S:Guidelines for the management of inflammatory bowel disease inadults. Gut 2011, 60: 571-607.

13. Bogdanos DP, Rigopoulou EI, Smyk DS, Roggenbuck D, Reinhold D,Forbes A, Laass MW, Conrad K: Diagnostic value, clinical utility andpathogenic significance of reactivity to the molecular targets of Crohn’sdisease specific-pancreatic autoantibodies. Autoimmun Rev 2011, 11:143-148.

14. Roggenbuck D, Hausdorf G, Martinez-Gamboa L, Reinhold D, Buttner T, Jungblut PR, Porstmann T, Laass MW, Henker J, Buning C, Feist E,Conrad K: Identification of GP2, the major zymogen granule membraneglycoprotein, as the autoantigen of pancreatic antibodies in Crohn’sdisease. Gut 2009, 58: 1620-1628.

15. Op De Beeck K, Vermeire S, Rutgeerts P, Bossuyt X: Antibodies to GP2,the major zymogen granule membrane glycoprotein, in inflammatorybowel diseases. Gut 2012, 61: 162-164.

16. Joossens S, Daperno M, Shums Z, Van SK, Goeken JA, Trapani C, NormanGL, Godefridis G, Claessens G, Pera A, Pierik M, Vermeire S, RutgeertsP, Bossuyt X: Interassay and interobserver variability in the detectionof anti-neutrophil cytoplasmic antibodies in patients with ulcerativecolitis. Clin Chem 2004, 50: 1422-1425.

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Authors

Antonella Radice, PhD

Johannes Schulte-Pelkum, PhD

Lucile Musset, PhD

Makoto Miyara, MD, PhD

Gabriella Lakos, MD, PhD

Carol Buchner, MT (ASCP)

Michael Mahler, PhD

Gary L. Norman, PhDMarcos Lopez-Hoyos, MD, PhD

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Anna Eslami

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