EUROIMMUN = EUROIMMUN | fiiisisiosn cos ad ANA Diagnostics Using Indirect ImmunofluorescenceEUROIMMUN Table of Contents Autoantibodies against cll nuclei (ANA). ‘Autoantibodies against cll nut, homogeneous. ‘Autoantibodies against dsDNA ‘Autoantibodies agsinst DFS70 patter. ‘Autoantibodies against nucleer membrane. ‘Autoantibodies agsinst nucleoplasm, coarse granular. ‘Autoantibodies ogonstnucleoplasm, fine granular Autoantibodies against Ku, ‘Autoantibodies against M2 ‘Autoantibodies against PMSal.... ‘Autoantibodies against RNA polymerase ‘Autoantibodies agoinst U2-nRNP /fibearn Autoantibodies against S70, Autoantibodies against nuciar dots Autoantibodies against contromeres ..- ‘Autoantibodies against PCNA. ‘Autoantibodies against controls. Autoantibodies against spindle fires. ‘Autoantibodies against midbody »... ‘Autoantibodies against mitochondti. Acad pet ovo eae ‘Autoantibodies against Jo-1 7 ‘Autoantibodies against SAP and PL-12. ‘Autoantibodies sgainstGolg apparatus. Autoantibodies agains ysosomes, Autoantibodies against Fa ‘Autoantiodios against vimentin Cytoplasmic ings & rods. Dilution scheme for immunofiuorescence EUROIMMUN Autoantibodies against cell nuclei (ANA) Definition ‘Antinuelear autoantibodies are ected ageinst antigens ofthe coll nucleus ‘Thoae autosntigens are named after ther biochemical characoristcs (ONA, histones, rbonucleoprotsins: RNP, the disease associated wih the core sponding autoantibody (SSA, $58: Sjogren's syndrome, antigens A and B; PMSc! polymyositis, progressive systemic sclross or, occasionally after the patontin whom the corresponding antibody was frst dotcted (Sm, Ro, La) ‘More than 100 autoantigens are presented in HEp-2 ells. ‘The mest important among them are Polyruclectides Double-strended DNA, single-stranded DNA, RNA Histones HI, HZA, 2B, 3, He, HZAH28 complex Fibonucleoproteins Ut {nJANP, Sm, SSA (Ro, SSB (La) [Nudleolar antigens U3 n|RNPMrilarn, RNA polymerase, PM-Sel (PM, 7-2ANP [To], 48S-RNA, NOR 80 (nucleolar organiser centromeres _Kinetochore protsins Other proteins Sa-70, PCNA eytin 1), nuclear granules, Ku, Mi2, lamin, lamin ocoptors Anais ‘Duetoits high sensitivity and spect, the Indirect immunofuorescence test {HFT using humen epithelia cells (HEp-2} and primate livers the gold standard forthe detection of ant-nuclear autoantibodies (ANA), The signal intersitis of «positive anda negative sampled signiicandy and microscope valuation Allows an exact determination of how the indicator dye (usualy fuoresceal is Spread in the tasuo or calls. Each bound autoantibody eauses atypical fuo rescene pattern, depending on th lation of the conespondling autoansgen ‘ifthe analysis result is postive, test substrats with defined single antigens {ELISA westorn blot, ine blot aroused for further differentition. Using mo ‘ospoifie test methods alone Is not sufclont fr the determination of ant- ‘clear autoantibodies since not all rlovantartigensare availabe thet pur fied form. For varfcaton of analysis results, monospeci tests should alwsys ’be accompanied by IFT.Evaluation [Ant-nuclear autoantibodies in patent serum are characteristic in many die- ‘aes, in particular, but not exclusively, rheumatic dseeses Inthe foreground aretha folowing: ‘Autoimmune diseste ‘Systemic lupus erythematosus (SLE) Drug-induced lupus erythematosus Mixed collective issue sisoase(MCTD, Sharp synde:) houmstid arthritis Other thaumatcdisasos Progressive systemic slrosis Polymyostis/ dermatomyositis Sjogren's syndrome ‘Autoimmune hapattis Ulcerative colts ‘The dotuetion of autoantibodies againt coll nc is an important diagnostic Indlestor in many autoimmune diseases. Antibodies against cles antigens are directo against various cll nuclear comporents (biochemical substencas inthe cell njceus). These encompass nucle acids, coll nuclear proteins and "ibonuelooprotsins. They ae acharactrstie finding in meny disease, inp teular rheumatic diseases. Tho frequency (provalance) of at-aucearantbod- les in Inflammatory theumatc diseases Is between 20% and 100%, and iti lowostin shaumtes rth et between 20% and 40%. Therefore, diferent antibody iagnostics against aucear antigens isindispensibo forthe agnosis of individual rheumatic dseeses and thai dferntation from ather autem ‘mune dseates ‘Systemic nus ervthematosus “Tho atermination of antibodies aginst double-stranded DNA (dsDNA) is con ‘Sideed tho most Important ertorion forthe dlagnosis of systemic lupus ery ‘thematosus (SLE) Immune complexes consisting of dsDNA and corresponding ‘utoantiodtes cause tissue damage in the subcuts kidneys and other organs. ‘The antibody ter correlates with te activity ofthe disease. Antibodies against ‘uelecsomes and Sm are also considered to be pathognomonic for sutosnt EUROIMMUN dias in SLE, Antibadis agsnst other polynuctetis, rbooucaotdes, his- tones and further nuclear antigens canals be datectd in this cisease. In drug-induced lupus erythematosus with manifestations suchas arhalga, ‘hits, exenthems, sores’, myalgia, hptomegalia and splanomogalia, 3n- ‘bodies against histones are aways observed, This reversible form of SLE can ‘ae induced by antibatis (e.penillin, streptomycin, ttracyelines),charo- therapeutic agents e.g. INH, sulfonamides), anticonvuisants leg. phenytoin hydantoies|,antarrythmics (e.g. proesinamide, praccololl, anshyperensives (e.g reserpine, hydralazine), psychotropic (eg. chlorpromazine), antithyroid rugs (eg. thiuracl derivatives), ant-vhaumaioid basis therapeutics le. {geld peniclamine) and other drugs such as contraceptives and allopurinol, ‘Autoantibodies in systemic lupus erythematosus Antigen Double-stranded ONA Singl-sranced DNA Nucleosomes| NA. ANA helicase A Histones Utne ‘sm SSA (Ro) SSBiLe) PCNA even) ku FitosomslP proteins {Hsp-90: heat shock protein, s0%Da (ea ‘Mixed connective tisue disease High autoantibody ters aginst UT-nANP ate charactors for mixed connae tive tissue disease (MTD, Sharp syndrome). The antibody ter coreates with the activity ofthe dsoasoEUROIMMUN _EUROIMMUN: ‘Autoantibodies in mixed connective tissue disease Antigen Un-aRNP Single-stended DNA ‘Bhoumatod arthritis In theuratoié ath, antibodies against histones can be observed in upto half ofall cases, whereas antibodies aginst Ut-nRNP are found more rarely. “Antbodios agsinst RANA ("rheumatoid arthritis nuclear entigan"} cannot be ‘etorted using HEp2 cel ‘Anté-nuclear antibodies in rheumatoid artis Antigen Histones ‘Singlestranded DNA Ur-nRNP (RANA, Progressive systemic sclerosis Progressive systemic selersis (PSS, scleroderma) can manifest set in two forms, which cannot always be clearly diferentisted. Until now, antibodies ‘gains fibilarin, RNA polymerase and Sc-70 havo only been observedin the ‘ffs form of the dlisesse, Autoantibodies against contvomeres ee associat ‘withthe limited form of PSS. ‘Autoantibodies in progressive systemic sleosis limited form) Pravalones 80-95 ‘Autoantibodies in progressive systemic selerosis dius form) Antigen Prevalnce (%) ‘Sa70 25-15 NA polymerase 4 ku, ine. overlap syndrome 25-80 TANF To) rare [NOR (nucleolar organisa region) rare Polumvosits dermatomyositis ‘Autoantbodios against PM-Se occur in polymyositis and dermatomyosis Other ant-nucear antibodies (Mit, Mi:2 and Ku) and antibodies against Jo-1 ‘ean also be found in these diseases ‘Autoantibodies in polymyositis and dermatomyositis Antigen PPM-Scl(PW-1, inc. overlap syndrome with PSS Jo-t hist -RINA syothetse) i Mz Ku, nc. overlap syndome Singlestrandod ONA PLT [threonylNA synthetase) PL? lalanylARNA synthetase) ‘Sigren's syndrome In ovimary!Siogren's syndrome, ativodies egsinst SSA and SSB are pre- ‘sent, mainly in combination with one another. In additon, autoantibodies against the salivary secretory ducts are found in 40 to 60% of cases. ‘Autoantodios in progressive systemic slarosi difluee form) ‘Antigen Prevalence () Fibrin 5-10 MSc! [PNC1}: [75KDa/1004Da in antigen) 18077) ‘Autoantibodies in primary Sidoren’s syndrome Antigen Prevalence (6) $5.4 (Ro) 40-95 3855 (La) 40-95EUROIMMUN EUROIMMUN ‘Autoantibodies in primary Siogren’s syndrome Antigon Prevalence () ‘Single-tranded DNA 13 (RANA 70) (Salivary excretory ducts 40-60), {Rheumatoid factors + 60-80), Primaria cithosis In edition to antibodies against mitochondria, various autoantibodies against call muce are associat with primary biliary irhosis, Some of them ean be ‘oneidered pathognamanie. Furthermore, antibodies against SS-A and con- tuomeres can also be frequently found in PAC. The presence of these two ant- bodies or antibodies against GP2T0 indicate sn unfavourable prognosis ‘Antinuelesr autoantibodies in primary biliary cithosis Antigen Provalence 6) Nuclear dots 25-40 Nucleer membrane 20-40 SSA 2 Centromeres 20-30 ‘At times, antibodies against nuclear antigens are detectable in subjectively lthy individual, with a prvalence of 5% and usually at low iter (ferent immunoglobulin classes, but maint la. ‘Antinuclesr autoantibodies: The most important associated diseases Antigen Disease Prevalenes SONA __Systemiclupus erythematosus (SLE)___———60-80 SLE 70-95 Druginduced SLE © [MCTD (Sharp syndrome) 20-50 Polymyositsi dermatomyositis 0-50 Progessive systemic sclerosis (PSS), ‘Sjogren's syndrome, rheum. ets au Sue PSS, Sigren’s syndrome ssDNA Antigen Disease Prevalence (%) Drug induced SLE Histones Ste Rhoumatoid athe [MCTD (Sharp syndrome] UnaRNP Sue - houmstois arthritis = Sjogren's syndrome Sue 55-8 Ro} Neonatal lupus syndrome Sjogren's syndrome SSB (La) Fiorilarin RNA polymerase | “RNAbelicase A PMscl Pmt) ‘centromeres Ply idrmatomvositie/overlap syndr. SS, fuse form PS; ited form ‘Scl70_____ SS, difuso form (yelin PCNA) SLE a SLE Ke __Paly-idermatomyosits, PSS Mi, ME2——_Dormaromyosits ‘Antibodies agsinst cytoplasmic components of HEp-2 ells cannot always be Cleary difereniated by thet immunofuoressence peter. Only 3 few cyt plasm reactive antibodies ran be assigned ta particular disease, 9. antibod Tes against mitochondria in primary bila crhosis and antibodies against the proteins, Jot, PL-7 and PL12 in polymyositis and dermatomyositis. Further Fare antibodies found in polymyositis are those directed against QJ, EJ and ‘Signal recognition patos (SRP. Oiher cytoplasmic antibodies ~ against ivo- ‘somes, Golgi apparatus, iysosomes and cytoskeletal components such as vi ‘mentin and yfoterating are of minoc clinical signicenca. The ciagnesti val Us of mitsis-essorated antigens has also not yet been frally clarified When allthese arguments are considered the high immunological relevance andthe resulting diagnostic value of anti-nuclear autoantibodies becomes dearEUROIMMUN {i a st cell nuclei, homogeneous HEp2 cells, Primate iver Hep-2 cella show # homogenous fuorescence of the cll nucleus, The con ddoneed chromosomes of mittc calls are eccentuated. The ares surrounding the enromosomes dark (nthe substrate primate liver 3 homegensous, pay coarse to fine clumpy fluorescence ofthe cll nucle ean be observed, Known target antigens: dsDNA, ssDNA, nucleosomes and histones, EUROIMMUN Autoantibodies against dsDNA [AntidsDNA positive AntidsDNA negative AntisDNA nogative (Winetoplast) (coll mucus} (basal body) ‘The standard substrate forthe immunofluorescence tet Is the haeroflagel late Grthafa ucla. This possesses a dsDNA containing giant mitochondrion (kinetolast! which, apart from dsDNA, essentially displays no antigens which cour aga inthe coll nucleus. Antibodies which react wit the Kinetopast aro therefore directed exclusively at Gs0NA. With C uci they produce a ham” genous, party edge-accontuatd fuoroscenco ofthe Kinetoplat. Any reaction inthe call puctous is nat evaluate: uorescance in the basal body of he lage [ims without significance. Antibodies to #s0NA cannot stain the Knetopast Cnical association: Autoantibodies ageinst dsONA are found exclusively in SLE and in 40-00% of cases, depending on the method of investigation and tha Alisease activity.HEp-2 calls (On the substrate HEp-2 calls autoantibodies ageinst the DFS70 enigen dept ' homegeneous nuclear pattern of dente fine speckoe with» granular fuores tence of the comatin in the metaphase Known target antigen: DFS70 or LEDGF len entheium derived growth facta] Clinical association: Autoanibocies against DFS70 have been found in patents with liferent ceases, euch ae stops dermatiis, asthma and intratial ers ties, and in heslhy Blood donors. Due to thelr low prevalence in systemic eu toimmune rheumatic diseases ithad been diacuered whether the detction of ‘these autoantibodies can be used as an exclusion criterion. thas recently boon shown, however, that anti-DFS70 antibodies also occur in rheumatic daeases with 3 prevalence of upto 17% The clinical association fs therefore uncles. EUROIMMUN aoe Autoantibo s against nuclear membrane HEp-2 calls Primate ver (On HEp2 cells the interphase ells show a homogeneous fuorescence of tha rucieus, with the rim ofthe nucius acentuated, The chromosomes of mitot ols aro dark Cn tissue sections of primate liver a characteristic linear Suorescence of the rnuclear membrane can be 20, Known target antigen: GP210, lamin A, lamin B, lamin , lamin B receptor Cinicalassocition: Antibodies against nucle Dilarycletosis (PEC), ‘membrane occu in primary= EUROIMMUN — {ersstncrns —_— — EUROIMMUN {ssh Autoantibodies against nucleoplasm, coarse granular Autoantibodies against nucleoplasm, fine granular Hep cells Primate liver HEp2 eels Primate liver HEp.2 calls generally show a coarse granular, sometimes medium tofine oran- aegis aso sey te fcr of Weal me Oe Tr ular fugrescence, whichis spreed over tho entire call nuclous leaving the ru phase, The nucleai are alee reactive, but they are sight shouetied against Cleo trea. n mito cols the condonsed chromosomes ore dark while the aryopasm In some samples they do not react tall Mat al show @ perphary shows an almost homoganeous, smooth fuoessence. Granular orescence, nth the chromosome excised, Tissue sections of primate liver slee show a granul orescence. The nec (n tssue sections of primate ver thre is no granular reaction inthe hepate- do notreac. The anbodies rest wth primate ver tothe same extant a with tem, bute neta show asmooth Nuorescancsinsempes wih a high Hep cal antibody ter. | Known target antigens: Ut-sANP and Sm. Known target antigens: SS-Aand SSB. Cini association: SLE and mined connective tisue iss | ctnct association: Sidgren's syndrome, SLE and neonatal LEEUROIMMUN Autoantibodies against Ku HEp-2 cells Primate fiver In the indivetimmunofuorescence test with HEp-2 ealls, antibodies ageinst Ku exhibit a fine-speckled fuorescence ofthe cal nuclei and the nucieo ara sitive in parts. itis ficult to recognise the diference to antibodies to SSA, 5538, Smand FNP, owover, if primate ver sections are incubsted in poral, possibly inthe same ed, 2 ypcel lumpy speckled staining of te call nil s found, which is almost carta prof of antibod to Ku. linia association: The provalence of autoantibodies against Kus 25-60% in ‘overlap syndrome of poly-dermatomyosits and progressive systemic slo sis [fton accompanied by primary pulmonsry hypertension, 5-10% in var ‘us forms of myositis, 10% eystemic lupus erythematosus and upto 3% in EUROIMMUN fitsisugossu a Autoantibodies against Mi-2 HEp2eolls Primate liver ‘Autoontitodies to Mi2 show a fine speckled fluorascance ofthe call nucle in ‘the indirect immunoflurescence text with HEp2 cell. The nucleo ae party Unaffected, ‘With primate liver autosnibodis against Mi2 dopet a fine-granular uores- cence ofthe hepatoeyte nucle nical association: Antibodies against Mi-2 are highly speelic merkos for dermatomyositis with ral fotdhypertephy. They re found in 15—30% of pe tients with dermatomyosita and in 812% of patients with idiopathic myositis,EUROIMMUN Autoantibodies against PM-Scl HEp-2 calls Primate liver inthe immunofluorescence tes with HEp2ells, autoantibodies against PM-Sal exhibita homogeneous fuorescence ofthe nucaol with a simultaneous weak 2, fine-specked reaction ofthe nucleoplasmn, The condense chromosomes of tha mitotic cals ae unaoctod: a ie, spectad Fuoresconce is shown outside ofthe chromosomes. A homogeneous luorescenc ofthe nucleoli alsa appears wth frozen sections of primate ver, a wall as avery week, ine-speckod to reteular staining ofthe eal nucle nica association: PM-Scl antibodies canbe detocted in 18% of patients with pelymyesiisystemic slerois overlap syndrome, Hero, the autoantibodios ‘are ustaly irectea against both main antigens: PM-Sel7S and PM.Sc100 progressive systemic seleosis is exclusively presen, antibodies to PM-Sci75 Show a prevalence of 10%, and antodies to PM-Sl100 a prevalence of 7% With tos sytoms which detect only ant:PMSeI100, some petients with pro rassive systemic slerosis romain unigentieg Bs am ot EUROIMMUN !ssissiune = Autoantibodies against RNA polymerase | Primate liver HEp2 calls HEp-2 calls show e granular fluorescence ofthe nucleoli The nucleopiasm i almost dark In mitotic calls the region of condensed chromasomes is not Stained. Outside ofthe chvomosomes a fine granular to smooth fluorescence fan be seen In mitotic ells one to several dots may foreste, which core spond tothe nucleolus arganieator (NOR} Cineat association: Antibociae against RNA polymerase! have ofr only been detected in progresive systemic selerosis (dite form). The prevalences 4%oe EUROIMMUN Autoantibodies against U3-nRNP/fibrillarin HEp2 cells Primate liver (On Hep-2 ells interphase calls show a granular fluorescence ofthe nuceal Mito calls have a coronary peichramasomal uorescence, ‘Tho substrate primate liver depicts a homogeneous fuorescence ofthe cal et Ctnical association: Antibodies agains ibilarin have so fat been observed only in progressive systemic scorsis eiffuse form). The provalnce is 5- 10%, HEp-2 cells Primate ver HEp-2 calls show en almost homogencous nuleat fluorescence in interphase 218 accentuated also showing e homogeneous fluorescence. mitotic calls only the border ara of condensed cho: calls The muck The eyiopasm is dark. rmosomes fuoreses. Clinieal association: Se1:70 antibodies are detected in 25-75% of patients with progessive syetomic sclerosis ifuse form), depending on the analysis meth fd and the activity of the teaseEUROIMMUN ae Autoantibodies against nuclear dots HEp2 cells Primate liver In immunofiuorescence using HEp2 eels, fv to more than twenty feral ‘Saad granules which are spread over the call nucleus (nuclear dot) can be ‘geoninthe nucle during intorpnose. The ovoplasmis dark antibodies against Inllochondia, which are associated with primary ilar ctrosi, are not pro ont atthe sama tm. In mitotic calle the nuceat dots are dissolved. Ousioe the unstained) ehromasomes only isolated granulo fueesce Antitodies against nuclear dots rect with primate ver othe same excent 35 ‘vith Hep? cells if both substrates ere used in parallel, tsa antibodies can ‘even be idened if anibacies against centromeres ae present atthe some time, This can occasionally be observed in eases of primary biliary chosis Known target antigens: Sp100,Sp140, PML, SUMO (lineal assoctation: Autoantibodies against nuclear dots occur in 10-30% of pationts with primary biliary cross. EUROIMMUN | {isis Autoantibodies against centromeres HEp-cells Primate liver HEp-2coll show voy secteuoresconce pattern, which is ceracteriaes by fine, evenly sized granules [generally 48 or 82 cantomeres par eal nuciusl ‘The granules in itorpaso calls are spread evenly over the nuclous, while In ritote calls they ate arranged either rbbonlike inthe madan plane (mot hase) or in two parallel ibbons approaching the centrioles (anaphesel, (n tissue sections of primate fiver 10 to 20 granules, which are spread over the cll nut, canbe Seen. The fluorescence ofthese granules Is lgnfesrily weaker than the HEp2 cal taining and i therfore easy to mss. Mitotic cll Bre only rarely detected on liver substrate Clinical association: With high specificity and a prevalance of 20-95%, an- tiboeies against centromeres are pathognomonic forthe limited form of pro restive systemie eterosi Inthe limited form the extremities are favoured 4nd the inner organs less affectedEUROIMMUN | {iso — Autoantibodies against PCNA HEp2 cll, Primate fiver Autoantinoses against PENA show a cell cyele-dependent fuorestence pattern with HEp-2 eal Hal ofthe cell nuce of ll interphase colle exhibit a bright Fine speckled basic uorescence with the nucleoli being unaffected. The same flooresence pattern i found withthe other haf, But the intensity slower by f factor of 10. Th area ofthe condensed chromasomes isnot stained inthe rritosis; the surounding rea ofthe chromosomes shows only @ weak, fine Speckled fuorescence, corresponding tothe darker nuclei of te interghase callin pattern and intensity. ‘The rection with primate Wve is largely nogative CGnieat association: PCNA antibodies or space or SLE, The prevalence, however, only 3%, EUROIMMUN i= Autoantibodies against centrioles HEp2 ols Primate ler ‘\rypial postive result is characterised by fluorescing centrioles inthe evo plasm of HEp2 calls, namely ane or two cantralos por cll Im mitotic ‘entiles ae located at two opposing poles, ‘With primate fiver, high ter samples produce smi fuoressing dats in the ‘topes of hepetocyes. ‘intalassocation: high ter > 1:1,000ineates progressive system rosis or Raynaud's syndrome. But the provatonceis ony afew percent.EUROIMMUN since Autoantibodies against spindle fibres Ps . e HEp2 calls Primate iver Using HEp2 calle antibodies against MSAT and MSA? (HSAGSI antigens can be detctod. MSA antibodies cause fine Granular to racular pattern ofthe sucloar matrix in ineephese ells. The nuceali do nt fluoresce. n the pros lence of MSA antibadis the interphase cell auce are not seine. On tissue sections of primate liver «granular fluorescence ofthe cell muti can be observes. nical association: Autoantibodies against MSA-1 are associated with Sjogren's syndrome, but they are aso found in other heumsticdseasos. Ato snibodies against MSA.2 occur predominant in SLE. EUROIMMUN Autoantibodies against midbody HEp-2 cells Primate fiver In the incest immunotluorescence test, HEp2 calls in the metaphese of mi tosis show 2 ine granular fluorescence ofthe median plane inthe presance of ‘midbocy antibodies. In contact tothe pattern found with antibodies to con Utomeres this Muorescing ine remains in the mie until the end of mitosis Ther length corresponds oe whole cel width inthe separatin zona, andthe line ineresingly shortens until ony 2 fluorecing dt is sean inthe telophese, binding the daughter cals together ("goodbye tse). Half of the iterphaes alls contain numerous coarser fluorescing drops; the remaining cells are dark. On tissue sections of primate ver theres an unspecific fuorescanee, Cfrical association: The diagnostic significance ofthese antibodies is sll unEUROIMMUN {3 Autoantibodies against mitochondria Hep 2 cals Primate liver HEp-2-alls contain the atigans M2, M3, MS and Mo; hore the antibodies pro dive a coarse granular fluorescence ofthe eytopasm which doesnot inluse ‘he nucious (previousy, the iowise PBC relevant nuclear dots also reacting ‘were wrongly suspected of bing tray mfachondia ‘The primate ter shows a granular fluoresence af he cytoplasm. The cll nu ele are dark. The rosction ofthe tissue s generally weaker than that of HEp-2 cela Clinical association: Autosnibodies against mitochondria can be detcted in various diseases. They often occur together with other autoantibodies, eg. wth utoaribodies againet call nee. Antibodies to mochondka are of pertcular Significance forthe diagnos of primary biliary eitrnoss (PBC) The prevalance isupto 98%. EUROIMMUN | {iii Autoantibodies against ribosomal P-proteins HEp-2 calls Primate liver Autoantibodies against ribosomal P-protein ceuse a smooth to fine granular staining of the cytoplasm wen using HED-2 calls asthe aubetat, Hepatocytes ofthe primate ier show afuoresconce ofthe ene surface with pathy escentuation. There io reaction wit owsiter samples, Cnica association: Autoantibodies against ribosomal Proteins ar a charac teristic maker for SLE. The prevalence i round 10%EUROIMMUN See's Autoantibodies against Jo-1 Hep-2 calls Primate iver Antibodies against Jo- show a fine granular to homogenous cytoplasmic fu fescence in the indirect immunofluoreacance test (IFT with HEp-2 cols. The tell nucel ago show dtnct sharp das in many cases, According to recent ndings, these enzymes ae nat solely localised inthe cytoplasm, but ae a found in the cll nucleus in some species (On tssue sections of primate liver the oytopasm fs only slightly stained. The uoresoance cannot be usa for diagnostics. Cnieal association: Antibodies against Jo-1 can be detected in polymyositis with» prevalence of 25-36%, They ae offen associated with other concurrent autoimmune cloeases suchas SLE, systomicslerosi, ints lung ross, Reynaud's syndrome and polysynovts, EUROIMMUN {itorsistrosnn Autoantibodies against SRP and PL-12 Hp cells HEp-2 cells Autoantibodies against SAP let) produce a mainly eytoplasmic, smooth to fine ‘ranlar fuarescence on HEp-2 call In mile cols the forescence Is per "hromosomally intensified, the chromosomes are una Autoantibodies against PL-12 (right show fine granular to homogenous cy- ‘oplesmic uorescence with HEp-2 eels The coll nucle also show cistinct tear dots in many cases, According to recent findings, Use enzymes are hat solely localized inthe cytoplasm, but reals found in thecal rucleus in nical association: Antibodies against SAP canbe found in polymyositis and ‘dermatomyositis in approx. 8% of cases. They are also makers for necrois- ing myopathy, a autoimmune disease that cfr from aalymyosis, butcan ‘manifest with skin changes typical for dermatomyositis. Antibodies aginst PL-12 occur in myositis with 2 prevalence of 3,EUROIMMUN | $ssoriisevinns = oe EUROIMMUN fessor Autoantibodies agi ist Golgi apparatus Autoantibodies against lysosomes Hep2cells Primate liver HEp2 cells ‘Autoentibodies agsinetGolg apparatus present inthe Indirect immunofuores (on HEp-2 ell abodes against ysosomes show a fine to medium or osteo cence on HEp-2 cals as reticlar-granular structures which ee in contac with ‘droplet shaped Muorescence of the cytoplasm, tha cll nucleus on one side. The cytoplasm ofthe hopatocyes is also stained In HEp2 oals which aon tho mitosis, the Golgi apparatus is oo largo exten (On eeu sections of primate ive Aispersed, Here the anbadies show na reaction. eres an unspeeefoorescence Cnieal association: The diagnostic significance of those antbodies is un- (On primate lve the cytoplasm of epatonyes is alo stained. known, They can oecaianally bo found in healthy individual Clinical association: Autoantibodis against Goll apparatus occur in diferent sutcimmane clseases, paiculry in SLE and Sjégrn’s syndrome. Detection of those antibodies haa itle relevance du other low disease spect,EUROIMMUN Eesti Autoantibodies against F-acti vsMa7 HEp2 colle Primete liver ‘Autosnibodies against actin cause a microfilamantous fluorescence pattern Using the cll ne VSMA7 (vascular smooth muse) (On HEp2 ells individual or several bunched fibre structures fluoresce. They are located primarily inte cytoplasm, but can also steteh aver thecal nice On tissue sections of primate liver there is 9 strong rection ofthe bile canal Clinical association: The determination of autoantibodies against Factin is Of particular significance forthe diagnosis of AIM (prevalone around 50%), the exclusion ofa combined liver dsoase (overlap ayndromsl and for delim tation of AIH agaiast alcohol or drug-induced eirmosis and other forms Of fone iver inflammation, such as vieuesinguced hepatitis, primary ilery liver eichasis (PBC! and primary sclerosing cholangitis (PSC EUROIMMUN Autoantibodies against vimentin HE p-2 calls Primate liver Antibodies against vimentin cause staining ofa fine net of fore Inthe ey: toplasm of HEp-2 cell. The net is parteularly dense near the eel nucleus Ih mitotic eels numerous ound fuorescing droplets can be een outside the dark chromosomes. These are probably condensed vimentin, On tissue sections of primate liver ther ean unspecitie uorescene. nical association: ts not known whether autoantibodies against vimentin have any diagnostic relevance. The same goes for other rare autoantibodies such as cytokeratin, ropomyesin,vineuln, teEUROIMMUN | linn Cytoplasmic rings & rods Hep calls Rings and rods area cytoplasmic patern on HEp-2 ells that has buen desribed ‘only recently. Those filamentous structures, which are expressed inal sages ‘ofthe cel cyte, present themeelved as rings, rods or loop ie assumed that the reaction iscrected against the autoantigen inosine monophosphate dehy rogenase 2(IMPDH2|(Seolig ta). Clinical association: The depicted patton was observed mainly in patants with hepatsC infections, pariulaly aftr treatment with interferon-alpha or riba itn (prevalonce 35%) EUROIMMUN {iia = lution scheme for immunofluorescence jon of serum samples in steps from 110 oF 1.2 (square root of 10). recommended for all EUROIMMUN immunotiuorescence test systems. The in dividual ditutons canbe esly setup without the nood for numerical acrobet ies (210,132,110, 1:20, 11000, st). Previously the precision was exaggerated by using quadratic dilution steps, On the other hand rating by a factor of 4 results into rough a framework For every test parametor there is 2 suitable stating dilution. n order to sim ply the test procadure and evelustion of els, two antibody etegories are ‘iferentiates at EUROINIMUN: Anitbodies of group ae alroadyclagnosteally Felovant at at ter of 110, while those of group! sta 1:10 The titers determined for each sample ao classified using the symbols (4) to -++44 The difering clinical signicance of antibody titers forth two groups is ready incorporated into this scheme | BBN sro | ae | se | 1220 | 11.00 13200| 1000 In order o make optimal vse ofthe grasteatetia of indirect immunofores once, expersin this area always test forthe majority of autoantibodies using two dilutions, foc the following reasonsEUROIMMUN = ching effect: In twa out of every 100 higher sera an untypicl reel with the stating daion, Some strongly positive sora even rect a a negative they AMA tkidnod Ant-Yo (cerebellum) Ez Ant-opid. bas ne tongue) ANCA (granulocytes) [Autoantbody masking: f unspecific antivaies or additions, visually dom nant autoanthoodes are prgent into high 8 concentration, they oan mask a ‘elevant antibody Saeco 1300 fo) fo oa) [Antiibosomes and antinucteoli | | ANA, homog. and anti-centromeres| EUROIMMUN eral of factor of 10 are used, its without further incubations than with stepwise teations. Titer estimation: When two dilutions witha iter an be determine for most postive [soo taba). Rosulte are obtinad one day oa Fwuoceseonco at 11900 AAD ter negative 1100 wook 11,000 moderate 13200 strong 110,000 J In contrast, tis not posible to quay positive real from a single diston: Ab show vor different abating behaviour depending on the avy. This Is tc by the parallel analysis Photomtiesystoms based on eylothemtcal ‘or fluorestonee are obsolete [ANA, homogeneous (titer 11000) | | ANA, homogeneous (titer 110,000)