Pitfalls of doing ANA immunofluorescence Can we define ”false positive”? Can we define ”false negative”? Can results be compared between labs? Amsterdam March 2011 AW 2011 Background: HEp-2 IIF Current literature and reports from laboratories on different IIF HEp-2 cell staining patterns is commoly use often rather primitive terms e.g. Homogeneous ANA Can not be Speckled ANA linked with Nucleolar ANA a disease Mitotic spindle staining phenotype! Cytoplasmic staining AW 2011 Proposed taxonomy of HEp-2 cell staining patterns elaborated in our EU CANTOR project 1998-2000 Membranous nuclear patterns: – Smooth membranous nuclear – Punctate membranous nuclear Nucleoplasmic patterns: – Homogeneous nucleoplasmic – Large speckled nucleoplasmic – Coarse speckled nucleoplasmic – Fine speckled nucleoplasmic – Fine grainy nucleoplasmic – Pleomorphic speckled (PCNA) – Centromere – Multiple nuclear dots – Coiled bodies (few nuclear dots) Nucleolar patterns: – Homogeneous nucleolar – Clumpy nucleolar – Punctate nucleolar Spindle apparatus patterns: – Centriole (centrosome) – Spindle pole (NuMa)(MSA-1) – Spindle fibre – Midbody (MSA-2) – CENP-F (MSA-3) Cytoplasmic patterns: – Diffuse cytoplasmic – Fine speckled cytoplasmic – Mitochondrial-like – Lysosomal-like – Golgi – Contact proteins – Vimentin-like Negative Undeterminable Wiik A. et al. J.Autoimmun. 2010 AW 2011 IIF staining patterns on HEp-2 cell substrate. Membranous nuclear patterns: – Smooth membranous nuclear – Punctate membranous nuclear Nucleoplasmic patterns: – Homogeneous nucleoplasmic pattern – Large speckled nucleoplasmic – Coarse speckled nucleoplasmic – Fine speckled nucleoplasmic – Fine grainy Scl-70 like nucleoplasmic – Pleomorphic speckled (antiPCNA) – Centromere – Multiple nuclear dots – Coiled bodies (few nuclear dots) Nucleolar patterns: – Homogeneous nucleolar – Clumpy nucleolar – Punctate nucleolar Spindle apparatus patterns: – Centriole (centrosome) – Spindle pole (NuMa)(MSA-1) – Spindle fibre – Midbody (MSA-2) – CENP-F (MSA-3) Cytoplasmic patterns: – Diffuse cytoplasmic – Fine speckled cytoplasmic – Mitochondrial-like – Lysosomal-like – Golgi-like – Contact proteins – Vimentin-like Negative Undeterminable l a c i n i l c e v s a n h g i e s s e h t h i t w f o s l n o i Al t a i ! c s o e ass featur d n a AW 2011 IIF versus different solid phase assays Membranous nuclear patterns: – Smooth membranous nuclear – Punctate membranous nuclear Nucleoplasmic patterns: – Homogeneous nucleoplasmic pattern – Large speckled nucleoplasmic – Coarse speckled nucleoplasmic – Fine speckled nucleoplasmic – Fine grainy Scl-70 like nucleoplasmic – Pleomorphic speckled (antiPCNA) – Centromere – Multiple nuclear dots – Coiled bodies (few nuclear dots) Nucleolar patterns: – Homogeneous nucleolar – Clumpy nucleolar – Punctate nucleolar Spindle apparatus patterns: – Centriole (centrosome) – Spindle pole (NuMa)(MSA-1) – Spindle fibre – Midbody (MSA-2) – CENP-F (MSA-3) Cytoplasmic patterns: – Diffuse cytoplasmic – Fine speckled cytoplasmic – Mitochondrial-like – Lysosomal-like – Golgi-like – Contact proteins – Vimentin-like Negative Undeterminable l a c i n i l c e v ! s a n h o e i t s e e a v i h a c t h o f s s o s n All tom a antige tibody d p n e e a h n m t i d y f n m s o r a t e s d et o e t d m a ! l e s o t b e s u i u t o q B n i n e n n e h a b c c t e t no ificity tine u c o e r p s r e h t by o AW 2011 Why use HEp-2 cells for the screening technique? – Only intact permeable cells contain all the relevant autoantigens in situ and cells can be seen in different stages of division. But the reactivity with autoantibodies depends on whether the right conformational state of the antigen has been preserved. – Morphological recognition of HEp-2 cell staining patterns using one good HEp-2 cell substrate is a natural talent of many people: The European multicenter study (CANTOR) proved that! – Autoantigen mixtures coated on solid phase supports (ELISA plates, beads, arrays etc.) are unsuited for recognition of a number of diagnostically important single autoantigens. AW 2011 Relative percentage of positive results using ELISA screen vs. IIF HEp-2cell screen HEp-2 cells: set at 100% ELISA An example: SSA, SSB, Scl70, CENP-B, U1RNP, U1RNP/Sm, Jo-1, histones, dsDNA, ribosomal P, PMScl, fibrillarin, PCNA, Mi-2 Some assays: additionally contain cell extract of HEp-2 cells Solid phase assays like ELISA thus contain a limited no. of autoantigens (10-14). But- these targets are wellknown for their clinical associations with inflammatory rheumatic diseases and therefore 70-80 % of sera that are positive for ANA by IIF test are also positive by ELISA screen.. Some exceptions: JCA, DM/PM, SSc AW 2011 Percentage of autoantigens in HEp-2 cell testing positive by screen ELISA Nuclear and cytoplasmic targets seen by IIF HEp-2 cell screen (set at 100%) Relative percentage of IIF nuclear and cytoplasmic targets detected by composite ELISA: (about 10-15%!) Many of these IIF staining patterns have well-known clinical associations! AW 2011 Screening for ANA using IIF technique versus composite solid phase assay SJS SLE SSC MCTD PM/DM JCA 98 95 90 dsDNA 75 75 U1RNP SSA/B 40 15 Each column represents IIF ANA positive sera The bar shows the % sera found positive by ELISA AW 2011 Indirect immunofluorescence Very sensitive Broad screening potential Fluorescence Clinically meaningful cut-off setting is crucial ! How do you determine such cut-off? F Fluorochrome conjugate ”ANA” HEp-2 cell AW 2011 Five main HEp-2 cellular regions Nuclear envelope Mitotic spindle Nucleoplasm Nucleoli Cytoplasm AW 2011 Computer-assisted project to attain an agreed HEp-2 cell nomenclature: the EU-supported CANTOR project (1998-2000) Aim: To attempt to bring order out of HEp-2 cell terminological and conceptual chaos – To harmonize existing nomenclature using previous terms supplemented by description of visual characteristics as illustrated on agreed reference images – To supplement terms with the exact location of the stained structure(s) when necessary – To set up a rational taxonomy for the terms Wiik et al. 2010, J Autoimmun AW 2011 Substrate and images used – All images had to be classified at two magnifications: 200 x and 400 x. – All slides derived from one carefully selected batch of HEp-2 cell slides from one provider. – All laboratories used the same conjugate specific for human γ-chains (DAKO, Glostrup, DK). – All laboratories used incident light illumination microscopes with objectives possessing high numerical apertures for bright conjugate excitation AW 2011 Initial harmonization of terms – The three expert centres digitized images found by routine testing and brought them to sessions where 5 experts first judged them with regard to photographic quality – Images that were assumed to represent prototype staining patterns were selected as accepted reference patterns for the CANTOR study – Positive and negative staining characteristics were noted down and gradually agreed upon as new proposed and accurate descriptions of each pattern for the study – Each pattern was given a name that did not overlap with any other terms used – among others stating the location of the staining if that was felt needed (nucleus, cytoplasm, mitotic spindle apparatus etc.) AW 2011 Example of reference image display Large speckled nucleoplasmic pattern (”nuclear matrix”) AW 2011 200 X 400 X Another example Coarse speckled nucleoplasmic pattern 200 X 400 X AW 2011 Two rather similar IIF patterns 400 x 400 x Meta-phase chromatin plate negative Smooth membranous nuclear AW 2011 Punctate membranous nuclear Example of textual help: Nuclear membrane staining patterns Smooth membranous staining pattern: – -A smooth homogeneous ring-like fluorescence of the nuclear membrane in interphase cells. -Some samples with strong fluorescence may give an impression of whole nuclear staining – -A similar pattern is seen in the telophase cells. -In metaphase cells the fluorescence is diffusely localized in the cytoplasm, and chromosomal material is unstained. Punctate membranous staining pattern: -A discontinuous punctate fluorescence along the nuclear membrane. -On focusing through the nucleus the punctate staining can be seen on the surface of the entire nucleus. -A similar pattern is seen in telophase. – -In metaphase the fluorescence is diffusely localized throughout the cytoplasm. -Some samples with strong antibodies may give an impression of whole nuclear staining. AW 2011 Local and merged data – All data from each classification and each participant in each laboratory were recorded on local computers using the DOORS software and later merged into one common database. – It was now possible to compare intra- and interobserver variability between persons, groups and laboratories, calculate and compare kappa values between individuals, groups and laboratories using perceptometric tools of the software. – Expert classifications earlier agreed on (facit list) served as the key answers AW 2011 Sessions in the CANTOR project Phases: Inexper: Experienced: Experts: Education 29 ref. Images 29 ref. images N.A. Baseline 40 images 40 images 40 images Training 1 45 images 45 images 45 images Training 2 45 images 45 images 45 images Training 3 45 images 45 images 45 images Exam 40 images 40 images 40 images Between introduction of reference images, baseline test and the 3 training sessions graphic 2011 and statistical tools were used to illustrate results measured as a mean against theAW experts. Learning effect by computer-assisted Delphi round training with software. Kappa 1.00 100 95 0.90 Kappa value at start of course 0.80 Kappa value at end of course 74 0.70 12 Participants T E E T E T T N T N T T= trained, E= experienced, Non-experienced N AW 2011 Homogeneous nucleoplasmic pattern Reflex tests Lab.test: SLE, RA, JCA Chromatin constituents: dsDNA, histones, nucleosomes, HMGs Anti-dsDNA Anti-histone Anti-nucleosome Anti-HMG Farr assay, Crithidia IF ELISA Line IA SLE? ELISA Line IA DI-LE? ELISA Line IA SLE? At present: no assay JCA? Note that the choice of assay technique for anti-dsDNA has a strong influence on the value for clinical interpretation and use!!! AW 2011 Pitfalls – Homogeneous: some staining shows pos., others neg. nucleoli. The latter was named ”quasi-homogeneous” recently. Both are associated with infl.rheum.dis. And are chromatin pos. Some staining is reminiscent of this with pos. chromatin plates, but gives a very fine dense speckled pattern, directed to LEDGF. Mariz et al. 2011. A+R. – Many classify anti-topo 1 (anti-Scl 70) as homogeneous with pos. nucleoli and pos. chromatin, though the pattern is fine grainy. AW 2011 Anti-LEDGF: dense fine speckled nucleoplasmic staining pattern Mostly healthy individuals No reflex test available yet AW 2011 Pitfalls ctd. – Large speckled staining is difficult to distinguish from coarse speckled, but the antigenic targets (hnRNPs, vs. spliceosomes) and the disease associations different. AW 2011 Coarse granular nucleoplasmic pattern MCTD, SLE Assemblyosome constituents: Smith antigen U1RNP Reflex tests: line-blot, ELISA, haemagglutination AW 2011 Fine granular nucleoplasmic p. Sjögren’s syndrome, congenital heart block, SLE, dermatomyositis, healthy individuals SSA/Ro, SSB/La, Mi-2? LEDGF? Reflex tests: ELISA, line blot Reflex tests: RI Precipit. At present no assay AW 2011 Anti-Mi-2 antibodies: intermediate - fine speckled Dermatomyositis Reflex testing: Radio-immunoprecipitation AW 2011 Control line: IgG SmB SmD RNP-70 RNP-A RNP-C Ro 52 SSA/Ro 60 SSB/La CENP-B Scl-70 Jo-1 Ribo P Histones Conjugate Serum + conjugate Line immuno-assay AW 2011 Some examples of HEp-2 cell staining patterns and their most likely relationship to cell biochemistry and diagnostic entities Pattern Disease Biochemistry Most likely not recognized in a solid phase presentation of mixed autoantigens AW 2011 Smooth membranous pattern SLE,sero-negative RA Sjögren’s syndrome Anti-phospholipid syndrome Lamins ABC Integral membrane proteins Note chromosomes AW 2011 Punctate membranous pattern Primary biliary cirrhosis Nuclear pore complexes AW 2011 Pleomorphic nuclear pattern (PCNA) SLE, SjS DNA polymerase delta auxiliary protein AW 2011 Multiple nuclear dots Primary biliary cirrhosis SLE PML* body constituents: Sp-100, PML protein, 56K * Pro-myelocytic leukemia AW 2011 Spindle fibre pattern SLE HsEg 5 AW 2011 CENP-F pattern Malignancies (breast, lung, NHL, > 50 %.) Centromere protein F Note zipperlike staining Note different staining intensity AW 2011 Less common staining patterns (Examples of ”esoteric antibodies”) -Antibodies to Golgi complex: 6 known antigens Indicate SjS 60%, SLE 20 % Ataxia 5% -Antibodies to GW bodies: 8 known antigens Indicate SjS 40%, Ataxia 35%, PBC 10% -PCNA antibodies: Indicate SLE 40%, other autoimmunity 60% -Early endosome (EEA-1) antibodies: Indicate Ataxia 30% -CENP-F antibodies: 1 known large 367 kDa molecule (mitosin) Indicate malignancies: 50 - 70% (breast, lung, NHL) -Centrosome antibodies: 6 known antigens Indicate SjS, SLE. -Intracellular exosome antibodies: 7 known antigens Indicate polymyositis/scleroderma overlap, Scleroderma, RA -Nuclear envelope antibodies: 5 known antigens Indicate non-erosive RA, SLE, SjS, CAH AW 2011 ”Negative” Please note that: -A negative ANA result does not exclude presence of SLE or another autoimmune disease! -Anti-SSA/Ro and anti-Jo-1 are often not seen by use of several HEp-2 cell substrates (depends on fixation technique)! -Positive cytoplasmic staining is often called a ”negative result”! AW 2011 What are the clinical aspects? – Some ANA have well-known clinical associations, but the target antigen specificity needs to be revealed by techniques other than IIF (ELISA, bead assays, chip assays, immunodiffusion etc). – Some ANA have less clear-cut clinical utility, mainly because only modest efforts have been spent to harmonize their recognition by IIF and study their antigen specificity by independent techniques, and thus sufficiently large populations of patients have not been available for detailed clinical analyses. – Some ANA are very rare [”esoteric”] (<5%) and thus have not been focused on because they were considered clinically ”insignificant” although there is no basis for this assumption. – The present concept is that all ANA have clinically significant associations when large cohorts are studied, but that demands set up of co-ordinated multi-centre studies. AW 2011 Autoantibody conundrum: clinical value of esoteric autoantibodies. Studies of disease cohorts indicate low frequency (<5%) of antibodies to CENP-F, PCNA, NuMA, HsEG5, GW bodies, Golgi, early endosomes (EEA-1), PML bodies, coiled bodies ButStudies of serological cohorts of positive sera show a high frequency of certain autoimmune syndromes e.g.: Antibodies to PCNA, NuMA, HsEg5, GW, Golgi, EEA-1 indicate presence of SLE or SjS PML antibodies indicate PBC in 35% of cases! Anti-CENP-F indicates malignancies in 50-80% of cases! Antibodies to Golgi, GW bodies, early endosomes indicate ataxia in a high percentage of cases. AW 2011 Phenotypes of SLE: relation to serum autoantibodies Anti-ribosomal RNP: active systemic lupus with CNS involvement Anti-PCNA: Mostly SLE without a known phenotype Anti-spindle fibre: Mostly SLE or SjS without a known phenotype Anti-phospholipid: Mostly SLE with a propensity to develop arterial or venous thromobosis Anti-SSA/B: active cutaneous lupus, often with secondary SjS Anti-U1RNP: systemic lupus with myositis, RP, ILD and/or overlap SSc Anti-dsDNA: active systemic lupus, mostly with anaemia, nephritis Anti-C1q: active systemic lupus, mostly with lupus nephritis AW 2011 Clinical phenotypes of myositis: from antibody to most likely manifestations Fine speckled nucleoplasmic (Mi-2): clinical signs of proximal myositis plus skin abnormalities compatible with dermato-myositis, histological myositis, increased mm. enzymes and typical electro-myographic changes. – Coarse speckled nucleoplasmic (U1RNP): clinical signs of proximal myositis, histological and electro- myographic findings compatible with polymyositis, increased mm. enzymes, often signs of overlap myositis. – Homogeneous nucleolar (PM/Scl): clinical signs of proximal myositis, histological and electro-myographic findings compatible with polymyositis, increased mm. enzymes. Sometimes also signs of scleroderma overlap myositis syndrome. – Diffuse cytoplasmic (Jo-1, PL-7, PL-12 and others): clinical signs of proximal myositis, histological and electr-myographic findings compatible with polymyositis, increased mm. Enzymes, and often anti-synthetase syndrome (Raynaud’s, mechanic’s hands, interstitial lung disease, arthritis. – Mitochondrial-like cytoplasmic (SRP): Proximal and distal myositis with histological necrotizing myositis and electro-myographic changes. – AW 2011 Clinical phenotypes in scleroderma: from manifestations to autoantibody. 5 year survival: – Limited SSc with anti-centromere Ab.: 86% CREST symptoms, digital ulcers/digital loss, pulmonary hypertension, primary biliary cirrhosis, Caucasians. – Limited SSc with anti-Th/To Ab.: 65% Puffy fingers, intestinal involvement, pulmonary hypertension, often associated with hypothyroidism. – Overlap SSc with anti-PM/Scl. Ab.: 92% Limited skin disease, polymyositis, calcinosis, digital ulcers. – Overlap SSc with anti-U1RNP Ab.: 95% Limited SSc, polymyositis, pulmonary fibrosis, pulmonary hypertension, cardial involvement. – Diffuse SSc with anti-Scl-70 Ab.: 80% pulmonary fibrosis, tendon rubs, digital ischemia, sometimes heart and kidney involvement. – Diffuse SSc with anti-U3-RNP Ab.: 77% Digital ulcers, pulmonary hypertension, pulmonary fibrosis, African-Americans – Diffuse SSc with anti-RNA polymerase Abs.: 90% acute onset, renal crisis, tendon rubs, arthritis, arterial hypertension AW 2011 Conclusions. – ANA likely reflect tissue lesion mechanisms, genetic influences, and perhaps etiology, – ANA are linked to diagnosis, subsyndrome/ phenotype, manifestations, and prognosis, – May help planning of follow-up and therapy, – Have particular value in early disease forms, – Can best be revealed by IIF HEp-2 assay, – Many ”esoteric auto-Abs” are important, – ANA can be interpreted by many technicians, – Optimal use of ANA results depends on a close collaboration between clinics and labs. AW 2011 AW 2011 Autoantibodies to citrullinated proteins (ACPA) and diagnosis of patients with rheumatoid arthritis (RA): Genetic, clinical, technical, and epidemiologic aspects AW 2011 Early synovitis in RA? AW 2011 Clinical aspects: RA – The disease progresses quickly from a predominantly immmunoinflammatory to a destructive phase where established pannus erodes bone, tendons and joint capsule. – The ”therapeutic window” to get control of the early phase is very short (few months), and later conventional therapy has little or no effect on the destructive phase. AW 2011 Diagnostics of chronic inflammatory arthritides -Clinical history ¤ -Manifestations ¤ -Clinical examination ¤ -Radiological signs -Specialist evaluations -Histopathology -Immunopathology -Laboratory tests to look for inflammation -Immunoglobulin levels -Complement activation -Autoantibodies ¤ ¤ ¤ Clinical basis for setting a tentative diagnosis ¤ ¤ Clinical basis for setting a tentative prognosis AW 2011 Criteria-based early diagnostics Criteria = rigorously defined items Clinical symptom 3: rheumatoid nodules Clinical symptom 1: morning stiffness Clinical symptom 2: bone erosions? Not present Not found yet Specific serologic result: Anti-CCP Diagnosis and Prognosis Particular importance: clinical focus! AW 2011 Differential diagnostics in the clinic and the laboratory INF Healthy RA PsA OA SLE ASp Background Not really useful for differential diagnostics Differential diagnostic patients (signs, symptoms, simple biochemistry, radiology) Great importance for differential diagnostics AW 2011 Differential diagnostics RA Rheumatoid arthritis – Small joints – Symmetric arthritis – Rheumatoid nodules – Erosive lesions in joints – Rheumatoid factors – Anti-CCP (ACPA) PsA Psoriatic arthritis: – Larger joints – Asymmetric arthritis – No nodules – Bone destruction occur – Rarely rheumatoid factors – Anti-CCP (ACPA) rare AW 2011 Prevalence of Anti-CCP and IgM RF in some arthritic conditions INF RA PsA OA Healthy SLE ASp Background 1-2% 1-3% 2-10% 2-50%! 6-8% 70-80% 5-10% 70-80% 6-16% AntiCCP 5-10% IgM RF Sens. Spec. ~ 95%! 5-20% AW 2011 RF in some non-RA disorders – – – – – – Mixed Cryoglobulinaemia 100% Sjögren's syndrome 60-70% Systemic sclerosis 20–30% Systemic lupus erythematosus 15–35% Polymyositis/dermatomyositis 5–10% Many viral infections! hepatitis B, parvo B19, rubella, mosquitoborne alpha-viruses Healthy individuals 2–10%* * Depends on cut-off setting, age, and F/M ratio AW 2011 Citrullination of arginines in proteins taking place during cell death (apoptosis) H O H N O N peptidylarginine deiminase (PAD) Ca2+ NH H2N+ NH2 10 -5 mol. NH O NH2 L-arginine residue L-citrulline residue (+ charged) (neutral) Especially gly/arg - ser/arg repeat motifs are modified AW 2011 Cyclic Citrullinated Peptide: CCP An artificial ”mimotope” cfc1-cyc HQCHQESTXGRSRGRCGRSGS Cyclisation of the peptide enhances its recognition by RA autoantibodies Schellekens et al. Arthritis Rheum 2000, 43:155-163 AW 2011 How do synovial antigens become modified: arginine to citrullin? – Peptidyl-arginine deiminase enzymes (PAD2 and PAD4) are richly represented in monocytes, macrophages and neutrophils – When these cells undergo apoptosis Ca++ ions are permeating into the cells and activate PADs – Ca++ concentration in normal cells ~10-7M – Threshold for PAD enzyme activity ~10-5M – PAD enzymes most likely also act on the enzyme-containing cells themselves (Monos,MФs,PMNs) AW 2011 Association between anti-CCP production and shared epitope HLA DR typing and anti-CCP2 antibodies were studied in 268 RA patients from an early arthritis clinic cohort in Leiden. Radiographic disease progression was measured over 4 years. Carriers of shared epitope DRB1 alleles were more commonly anti-CCP positive than non-carriers (OR 13.3) and also showed the most pronounced radiographic progression. (van Gaalen FA et al.:Arthritis Rheum 50:2113-2121,2004). Shared epitope-encoding alleles are associated with antiCCP production, not with RA as such! (Huizinga T et al.: Arthritis Rheum 52:3433-38, 2005). AW 2011 Anti-citrullin peptide antibodies, IgM and IgA rheumatoid factors can appear up to 10 to 18 years before the onset of clinical symptoms of RA!!! AW 2011 Production of anti-CCP in RA Stable phenotype Onset of first clinical symptoms Anti-CCP level Cut-off value U/ml Detection limit - 10 Diagnosis 0 10 Time (Years) AW 2011 Genes and environment: tobacco smoking A: Sero-positive RA B: Sero-negative RA Figure 2 Relative risk (RR) for development of rheumatoid arthritis (RA) in current smokers (with different numbers of copies (0-2) of the shared epitope (SE) of HLA-DR) compared with never smokers. (A) RR for seropositive RA and (B) RR for seronegative RA. These graphs are schematic representations of the original data from a case-control study of RA reported in reference 9. AW 2011 Klareskog, L et al. Ann Rheum Dis 2004;63:ii28-31ii Copyright ©2004 BMJ Publishing Group Ltd. Genetic and environmental factors in the development of RA Figure 3 Schematic outline of how aetiological studies as well as interventions in the pathways leading to rheumatoid arthritis (RA) should be undertaken before onset of clinical signs of RA. CCP, cyclic citrullinated peptide; RF, rheumatoid factor; SE shared epitope. Klareskog, L et al. Ann Rheum Dis 2004;63:ii28-31ii AW 2011 Copyright ©2004 BMJ Publishing Group Ltd. Risk factors in RA. Recent study in 515 Danish RA patients and 769 sex- and age-matched controls Risk factors in anti-CCP positive RA patients: -menarche at = or >15 years of age (OR 1.87) -tobacco smoking (both sexes): confirmed, both former and current smokers, dose dependent effect -coffee consumption > 10 cups/day (OR 2.75) -alcohol consumption > 15 drinks/week (OR 0.58) -moderately demanding exercise (OR 0.51) -pets as adult (ever) (OR 0.73) Pedersen M et al..: Arthritis Res Ther 2006 AW 2011 Risk factors in RA Risk factors in anti-CCP negative RA patients: The strongest risk factor was increased body mass index 10 years before the study: -obese (BMI = or >30 kg/m²) (OR 9.79*) -moderate (BMI 25-30 kg/m², OR 3.53*) *compared to underweight (<18.5 kg/m²) -menarche = or >15 years of age (OR 2.27) -pets (ever) (OR 0.65) -moderately demanding exercise (OR 0.69) Pedersen M et al.: Arthritis Res Ther, 2006 AW 2011 Studies done on anti-citrulline antibodies are difficult to compare The citr. antigens are very different The cut-offs used are different The RA populations studied are different The differential diagnostic populations studied for comparison with RA patients are different – Some studies include undifferentiated arthritis, palindromic syndrome, RF+JRA, RF+psoriatic arthritis etc. all of which may actually become RA. – – – – AW 2011 Nosographic sensitivity (sensitivity in RA patients) – Anti-CCP, anti-filaggrin and APF show very similar sentivities: - at diagnosis < 6 months: around 50% - at diagnosis 1 year: around 60% - at diagnosis >2 years: around 70% – AKA and anti-Sa: usually show lower sensitivity than the above methods AW 2011 Anti-CCP in RA Data collected from 154 studies between 2002 and June 2009. AR & T 2010 AW 2011 Sensitivity Cut-off setting between sera from RA patients and immuno-inflammatory disease controls. This study compares a RA population vs. differential diagnostic populations 75% If such cut-off setting has been done by the developer there is less need for a study of inhouse immuno-inflammatory disease controls. Chosen specificity 98% 90% Clinical diagnostics need high differential diagnostic specificity! 1 - specificity AW 2011 How do you choose the optimal assay? Use the same disease population as comparator for each assay! Sensitivity 100% Choice: blue assay! Since this has the highest sensitivity 77% 70% AUC high 65% 50% Chosen specificity AUC intermediate AUC low Test of 3 ACPA assays Clinical diagnostics need high differential diagnostic specificity! 98% 90% 1 - specificity AW 2011 Test of specificity and sensitivity in RA vs.controls using 3 different assays RA/control Specific. Sens: CCP2 CCP3 MCV Bizzaro 100/202 98.5 54-74 67 62 Coenen 102/196 95 76.2-77.0 75.5 65.7 Damjanovska 566/351 93.4 56.9 56.2 52.5 Dejaco 164/303 98.7 70.1 n.d. 53.7 Innala 210/102 98.0 80.4 78.5-79.0 69 Mutlu 93/83 98.8 57.0-62.2 60.2 29 Soos 119/118 95 74.8 n.d. 69.7 Van der 272/463 98.5 67.4-68.0 n.d. n.d. 92/463 98.7 61.6-67.4 58.1 n.d. 180/463 98.7 65.2-77.4 67.1 n.d. 69.2 66.1 57.4 Cruyssen Van der Cruyssen Van der Cruyssen Average: 97.3 AW 2011 Note: Controls are not identical and stratification only approximated! Comparison of ACPA in terms of positive and negative predictive values. Study: Pts/con: CCP 2 PPV: NPV: CCP 3 MCV RF PPV: NPV: PPV: NPV: PPV: NPV 1 124/158 95.2 73.1 92.3 70.5 2 133/165 89.7-91.4 87.1-89.1 76.7 88.2 3 86/90 92.6-96.7 72.9-78.7 91.9-94.9 74.4 4 70/88 91.7 84.7 90.6 87.2 5 170/135 95.5 66.8 6 120/170 87.7-96.2 76.6-78.3 7 170/309 91.1 86.3 66.0 8 119/118 97.6 74.4 90.0 9 123/39 95.8 10 201/424 67.1 79.0 64.0 80.0 Average: 91.1 78.4 84.9 AW 2011 79.8 80.4 81.5 75.9 75.3 80.0 93.7 92.6 90.9 73.6 80.3 72.4 82.0 69.9 77.9 79.5 84.7 58.6 79.9 78.8 80.2 74.0 79.2 61.7 77.8 87.2 77.4 78.1 96.1 56.3 Conclusions – Anti-citrullinated protein/peptide antibodies (ACPA) are very specific markers for RA, also useful for differential diagnostics towards other polyarthritides but small subpopulations of other arthritides are ACPA-pos. too! – Cyclic citrullinated peptide 2 (CCP2) acts as a sensitive artificial mimotope for ACPA antigens in solid phase assays. – Anti-CCP antibodies are present very early in disease, sometimes before inflammation biomarkers rise and before clinical onset of arthritis is recognized. – Anti-CCP levels can decrease somewhat with remission induction and increase a little with disease exacerbation, somewhat parallel to but smaller than RF changes. AW 2011 Conclusions ctd. – High levels of anti-CCP antibodies are prognostic for an erosive disease course, not only in adult RA. – Anti-CCP antibodies prevail in RA patients carrying the HLA-DR4 shared epitope, most of which are RF-positive. – Anti-CCP is found in about ¼ of RF-negative RA patients, and these patients run an erosive course. – Several environmental factors influence the onset of antiCCP positive RA (tobacco smoking, coffee consumption, body mass index, alcohol consumption, exercise). AW 2011