Colorzyme’ Patterns Associated with Antinuclear Antibody Detection = o : « te of © ees * @ & so& @ ¢* © *e t/b toes com : 2 i e % e 2tG st e Naceolr and SSA/RoHlomogeneousand SSARo_Cenlromere and SSA/Ro SsAiRo immunoAssociated with ANA Patterns Pater observes usual Herogananus| Species spect (and/or ssa peter using Hep £2000" Cn ao be ANA regane Fe speck or ANA egatne Fine Speck (sometimes ‘ech reir tarng a wel) Fine Specks, Homogeneous, Neceor ‘a Gye Depa specced coase specea ca cycte Dependant Specie with packing im Sting in cleavage from etwean dong te Typso antibody Dscase()n which (coxa, an) tes moter auntie Nuseosone orCiromatny SLE, upsnaicad LE stone Dried Le, SLE Near Membrane upd nepas sm Sth) ark ant for SLE ue igh evel a MCTD and rout deco ssa High preaence in Sjore's syne ses comple, lower sevance motor morte deans ose Somers syrarone, inyosts, Neorata Lupus ssa Hah prevalence in iors spare seca complex tower revaoce othr sa70 arr ante ror Powe arte andy for SLE cea sere ‘Seon some patents th ting connective CSU Np 59-100, MD, POC 95 Assocated wth Primary lata cues Nsphcowe Some assocuton wih raignances iby Untrown 80 (cain) Untrcw conten Soon 57-02% of ptents| eh ie for (CHEST) of Slrderma art Raye henateron urs Mca Slee Spice Nuclear Steer note Connective tse eas ‘sooth Nill Paya /Sclrosema overap Characters of antgnc ‘Aistone complex Diteen cases a stone Nucor Lami A 8, Cant sp2i0 Proteins 68,0, €.F ard 6 oped wt, U2 70 a, Aan protaes ompned wath RMP 60 40a proton congtexed 40 10a proton coretered tn RNA parse 100 ko pots ot ‘oposameasa | ‘ary proton to OMA aymerese 3 an ana ote angen 95-100 0a poten 400 ko proton nero 80 a proton the eal by Fa paymerase | NOR 90, Past (PWS, others? Rowen tests wed 0 cont specie stoay Aor CZusrg onus to, IA ELSA Usa SA forgp-210 Irmunectison (0), EUSA Ccontematary wth Hep 2004" stanng pater, an tn, SA 1p,cusa Confem by staining pater contrnes by staning pate FSASystemic Rheumatic Disease Identification of the antinuclear antibody (ANA) pattern remains a crucial step in the process of diagnosing the systemic rheumatic diseases. ANA patterns often give the clinician insight {nto which autoantibodies are present and indications of disease likelihood, Ithas now been over 60 years since the LE cell was first described by Hargraves (1). During this time, substantial improvements in physician awareness, diagnostics and treatments have increased the survival rate for lupus patients from a 5 year rate of only 50% to a 10 year rate of 290% @). There is growing evidence that the appearance of autoantibodies may precede the onset ofthe disease, often by many years. Early detection ofthese autoantibodies may offer the opportunity for earlier diagnosis and treatment, improving the length and quality of life forthe patient, Arecent study in the New England Journal of Medicine reported that, on average, at least one antinuclear antibody ‘as present 3 years prior to the clinical diagnosis of SLE being made and at the time of diagnosis there were often 3 autoantibodies present (3). The earliest antibodies detected were ANA‘, anti-SSA/Ro and anti-SSB/La antibodies. ‘These antibodies were readily detected and reported using the HEp-2000® slide based assay. ‘The HEp-2000® substrate is Immuno Concepts’ patented ANA substrate that has been consistently proven in independent studies to be superior to standard HEp-2 for the detection and identification of ANA’s (4-7). When the ‘unique SSA/Ro pattern is present on the HEp-2000® substrate the laboratory can immediately report the presence of anti-SSA/Ro antibodies to the clinician, potentially accelerating the correct diagnosis forthe patient. ‘The photographs and charts presented here are designed to give the laboratorian a comprehensive guide to pattern recognition, antigen specificity, and disease association, Some of the more recently described patterns do not have disease association but are included for educational value. References: | Hargraves, M, Richmond H.,et al, Presentation of two bone marrow components, the tart cell and the LE cell Mayo Clin Proc. 1948;27:25.28, 2 Alamanos,¥, Voulgari, PY, etal, Survival and mortality rates of systemic lupus erythematosus pati northwest Greece. Study of a 21-year incidence cohort. Rheumatology. 2003;42(9):1122-1128. ‘3 Arbuckle, M. R,, McClain, M.T. et al. Development of autoantibodies before the clinical onset of systemic lupus cenythematosus, New England Journal of Medicine, 2003;349(16) 1526-1538, 4. Pollock W, Toh BH, Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa RolSS-A. ].Clin Pathol, 1999;52:684-687. 5. Bossuyt X. Meurs L., et al, Screening for autoantibodies to SS-A/Ro by indirect immunofluorescence using HEp- 2000 cells. Ann Clin Biochem. 2000;37:216-219. 6. Fritzler Mj, Hanson C,, et al, Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human {60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal, 2002;16-103-108, 7. Bossuyt X, Frans J, et al. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem, 2004;50(12):2361-2369,Flow Chart ANA Pattern Confirmatory Testing & Disease Association ANA Positive ANA Negative Ite ANA s nagatve, but aisease i suspected, ‘est or specie autoatbodion Legend DIL. - Drug induced lupus _ SLE - Systemic lupus Positive Negative tissue disease 2 @ @ er syndrome a Immuno Concepts has taken care that the information and recommendations contained herein are accurate and compatible with current standards. Nevertheless, it is difficult to ensue that all ofthe information given is entirely accurate in all circumstances, Immuno Concepts disclaims any liability, loss or damage incurred as a consequence, dizectly or indirectly, of the use and application of any of the contents of this chart.Negative Feport as: ANA Negative Susported amigo spect: Circa signicance: None Follow testing: None reauted: Report ax: Homogeneous Suspected amiganspacicity: DNA, DNB. Histono, DNA Binding proteins, others? nical snitcance: 1ONA postive. marker antibody ean In 60% of SLE patents, Followup testing: Contin FON antibodios Feport as: Nuciear Membrane Suspected eniganspacicity: Nudsarlarwne thers? Circ sianifeance: Mey be Senn pation ith SLE, PA, autoimmune hepatts, Follow-yo testing: None requted: Report as: Nucleolar Siposted atin sec iocnotnes? ono Creal sgnifcance: Soon Inpatents with ayeteme Followup testing: None requted. Feport as: Nucieolar Suspected emiganspacicity: FRNA Polymerase, NOR 9, theca? Cina! snfeancn: Seen vos wth foe ‘clerodarme, Follow-up testing: None requted. Root Spaced Suspected antign speci ini onad Peete Cina steno Seon [patients with evalung reoumstic ‘Siocaces. lou speci tae "8 Roper as: Cortromore ‘Suspected antigen specify: CContromore proton A,B, or Ginical significance: Seen in 57— 96% of ations with he limited {erm of Scleroderma (CREST) Folowun tein: Nene eae prosonssof ACA Report as: Nucteotr, suspect RitosomalP ‘Suspected antigen specifiy Esso Pets POP Cnet signitcance: Seon in up 10 20% of patonts with SLE Follow testing: Confirm with antirbosomal spoctc assy. Roper as: Nuceolar Barner eaten Ciricalsigticance: Seen in patonts with palymyostis Esiroderma everan, Folowup testng: Nove requied” Repor as: Hornogeneous, Speckled, and Nucleol Suspected antigen specticiy: ‘Toposomerasel Cricalsignitcance “Toposomerase | postive ‘marker antibody seen in 15-20% of patents with scloroderns, Eclon-up toting: Cont by EMetoslesHEp-2000@ 200% 4 Hep-2000@ 400x158 ae Bee Pe e = Fepor as: Speckled Suspeciod antigo section: Sm, ORR atid eee Cnica significance: Sm postive ‘parker aribody sven in f-40% ot SLE patents. RNP postive - seen in high ters n patents wth MCTO snd SLE, low ters nother saasas, Followup testing: ENA tstng to no out spectie ENAS. Fepor as: Speckled Suspected antigen spacticiy: UI- NE others? Clinical significance: Sm postive - ‘marker aritbody seen in 4-40 of SLE patents. RNP postive seen in igh ters in patents with MCTO end SLE, low itera in ther diseases, Followup testing: Contim by ENA tesng Report as: Speckled Suspected antigen spacticy: SSARo, SSeILa others? Clinica signfcsnce: SSAMRO {and SSBiLa postive - seen in high percentage of patents wth prmary Sierer's synsrome, S040 of patients with SLE, Fellowup vesting: Conte ehteang, ns conn Report as: SSARo Suspected antigon spect Susgcte antign spucioy A distinct speckled ae nucleolar pattern seen in 10-20% of he Interphase ruc. Thece aa the Fyperexaessing col, The remaning 80.80% of tho itorphese ‘als may or mey not dervonstate Saning. Tho ehtomosome rgion tthe metaphase rmtcte cll regative Report as: SSAVRo Suspected antigen spactciy: SSARo Clinical significance: SSAMo postive ~ seen in 60-70% of patents with primary Sirens syndrome, 30-40% of Batts wh SLE, Folawap testing. This pater rr to bao a thades EWA esting fo ae te Brosoce fant to oor ERs Colorzyme’ Patt Associated wi Antil Mitosis Stages of Cell Division Metaohase Avachase Te debieatonaed Exar Se ee ee Cantorcfthe cel and tha Spiele ters generated ‘tom the cantons) attach to thecentromerse, Becnse the nucleer membrane has ‘SssoWved,nanchremesome Specie anigens reditrbuto to ater region ofthe eal Tech Support 800-251-5115 Speckled & Nuclootar 16 (SSASSSB on regular HEp2) Fepor as: Speckled and Nuclslar Suspected antigen specicty: SSAo, S86/ka others? Cirical significance: SSAVRo postive - S00 provous gttor.SSBiLa postive. acon In 060% of patents with primary Sigren's Syndrome and 108% of SLE paints Also soon in conuneton with SSA/Ro In pation with neonatal upus Felowvup testing: Confim by ENA testingterns with Antinuclear tibody Detection Interphase DNAzynthosis and chromazome replication ‘ceurs, Genres replesie, ‘Telophase Spindle desoles anc romosome specic antigens become ‘Sonsinedwrthin the ‘Shomaeomal marx as Shenciow merrane www. immunoco: Prophase tromosomes concen, ences migrate. ‘ppomie eras ofthe eat Nuclew memérane fragmonts Cytokinesis| Col vision ie compote, om tere ining SStaosic ang ak seosteomo ncepts.com Report es: Speckled, suspect PCNA, Suspected antigen specfcy: ONA poymerase 9 leyeln) Cinicalsigifcance: PCNA positive: ‘atkerartbody seen in 2-10% of SLE patente Followup testing: Confirm by ENA testing 'p80-colin (1-5 dots) 2 « eo & 22 Report es: Seckle mitotic spi so present Suspected angon spostiiy: Nocoar Mitote Apparatus Giiical significance: Sjigren's Sypeomo, PRC, ethers? Followeup testing: ENA testing tere out Spacic ENAS. Roport as: Atypical spocklod, Nido Suspected antigen speci: Nibedy Cirical significance: May be ‘sean a Ew percentage of patios with scleroderma Folloysup testing: None reavired, confirmed by staning patter, Np l(CENP F) Report as: Atypical specks, suspect NSpil Suspected antizen specifiy Cantiomore roten *F*, others? Clincalsignicanco: Unknown, ‘some asoeiation with ‘malignancies, Folowsup testing: None required: Mutple Nucoar Dot (NSP I Pc 88) Feo as: Atypical speckled ‘suspoct Multple Nucoar Dots Susoected antigen spect: 988-1004 proton ine signtcance: Sannin 27-48% of patents wah PBC. Foloyeup tosting: None requires, Report as: Atyical speckled, ‘suspoct Oot Suspected antigen specticiy: Cain Cirical significance: Unknown. Followeup tasting: None requires,‘Mixed ANA pattern Mixed ANA pattern 23 25 Moxed ANA pattern on HEp-2000" Repor as: Conte and SSAIRO ‘Suspected antigen spectcity: Contvmnete prataa A, 8, of C; SSAVRo nical signicance: See sigrticance for sch respective pat Followup test: SSAVRa confirmed by pattom. Cantremero contig By pattern. Mixed ANA pattern on HEp-2000" pont as: Homageneous and Centromere ‘Suspected antigen specticity- PONA,ONE Histone, DNA Bncing proteins, Centromere protein A Bloc, Cincal signicance: Soon in Systeme slorons pation with Pulmonary disses epor ae: Homageneous and Spected Mixed ANA patterns can be caused by autoantbodes to ‘Several cferententgens or by antbodes to an anigon located in-sevorsl dferem areas of the call nucle, Ttarng and appropriate confmetory {ating foreach pater 5 recommenced Moxed ANA pattorn on HE. 2000" pon as: Atypical specie or Cell Cye Debendent Speciled ond Homogeneaus ‘Suspected anigan speci: Unknown {or atyseal spoked pottery Seo Fermogenecus angen spect incl sapence Urine, ie dapensert spac Fernggnai pen orn Sonscance. Followup testing: ENA testing to rule out ic ENS. Mixed ANA pattern on HEp-2000" FRepar as: Spaced end SSiVRo ‘Suspected antigen spect: SmyUTANF SSAF0, SSBMa, others Cncl significance: Sea sigrticance for eth respoctve pattrn Followup testing: SSAVRo abodes confined By pattrn. Confirm speckled patton by ENA tostrg MNO and AMA 28 suspect Centriole 28 ‘Chromosomal Coating 31 Mixed ANA pattern on HEp-20008 Report es: Atypl speckled, suspect Maltole Nuclear Dots (NSpti and {yteplasmie specklng. Suspected antgon spect 05-100, HD proten,rtochondra. incl Signfeanco: Seon in pationts ‘wth pemary Bilary errhos, Folowap testing: Nove recited Reports: ANA Negative, ‘suspoct Contile Suspected antgon speci: Gentile proteins Clinical significance: Unknown, Folowap testing: Neve reuive Repor es: ANA Negative, Mitotic Spindle ae Suspected antigen spect: IMitot sprncies incl sigitlance: Sean in ‘valving thoumatiecisoases, Folovewo testing: Nove recut Report as: ANANegative, suspect Epfesomel Contes Rinbode Paton: Stains only the chrornosomal area ct metaphase cols Segteteanaen spect nica signiteance: Unknown. Followup testing: None required, Report as: ANA Negative, suspect Gal Suspected antigen specticy Gola rote Cinicalsignifcence: Seen in patient wth primary Siogrens Byncrome and SLE Followup testing: None foqured.‘suspect Jo-4 Report as: ANA Negative, yeplamie sens Suspactes antigen speci Jor tity ANA Stas) Cinicalsnicance: Seen in pte 359% of pales with rest Fotloneup testing: Cantir with “oo specitetsting Roper a: ANA Negative, cytoplasmic specking ‘Suspoctod anton specifiy: EEA, GWig2, GW2, G3, Goods, RAPSE proteins Clinical stgnicance: Unknown. Followup testing: None requved: Repor as: ANA Negative, suspect Mtochovetial ‘Suspected angen specifiy: iMitochoneta Cnicalsipnitcance: Sn in up 10 80% of patents with PBC. Follow-up testing: Confim on AMA spect substete Such 35 Foent ties, opon a: ANA Negative, Sidroct Coste ‘Suspected antigen specitcty: ‘ubuln,vimentn, others? lnc signicance: Seen in aun eh sromenne vr Follow-up testi: Confim on antigen spectic substrate such as fodant seve, Suspected eniganspecticity: Retin Cnical sgnieance: Sean in patents haven har Follow-up testing Confirm on art spect substate such ae radent teeve Damage tothe substrate Caused by moving he vera o by touch soScebvaetaeh Poot Non-specific film caused by ‘adsorption of serum protens forthe sido ertac. Patter: Postve eaining of the knetopst inthe Chia lusiae Repor as: nDNA posite ‘Suspacted antigen spec “nDNA or dsDh ae Clinical sinicance: Marker ‘nibody soon it 30-70% of SLE patots Patton: No apparent staining of. the netopiat the Cath lsiae Report as: nDNA negative Suspected antigen specifiy None. port ag: Homagenaous and Speckle Suspected antigen spacticy. Lene eothelurrderived grows fectafattuse fine spaced WEDGFIDFS Cieical significance: Unknown, Follow-up testing: None recited "Wyn stating as boon oor shan sede. Whe ts saree opines cane petersKohler Illumination ‘Use Kohler Hlumination to optimize the ilumination of your sample and to reduce the visibility of the light source im- age. Flere’s how to do it Tar on the bright il light souree. Place a bite filer and a neutral density filter on top ofthe light source Place the slide onthe stage and set magnification to 1x or 20x. Focus the specimen, Close the field diaphragm as far as possible. This is located towards the bottom ofthe microscope where the light soure is coming from, Close the condenser o ris aperture, located under the stage, unt the fel of view outside the field diaphragm is evenly dark. Slowly turn the condenser focus knots to being the edges of the field diaphragm into the bes focus. ‘When the edges ofthe diaphragm are sharply defined, the diaphragm can then be centered. Use the condenser-centering screws to center the image ofthe closed field diaphvagen into the center ofthe field of view, 9. Open the felt diaphragm so thatthe edges are jut outside and beyond the fel of view. 10. Open the condense ors aperture to introdkice the proper amount of light and contrast 11. Adjust the light intensity as necessary. sees eum (lf reading COLORZYME®, a blue filter with a neutral density filter are suggested) Suggested Reading Slide Based ANA methods Kroshinsy,D, Stone JH, Bloch DB, Sepehr A. “Case records ofthe Massachusetts General Hospital Case 5.2009, 47-year-old woman with arash and numbness and pain in the lep.“N Engl} Med 2009;360(7): 711-20. ‘Moroni P, Schur PHL ANA screening: an old eet with new recommendations, Ann Rhourn Dis, Aug 2010;69(9) 1420-1422, Frtzlor Mj, Wall W, Gohl et al-The Detection of Autoantibotes on HEp-2 Cels Using an indirect immenoporondase Kit (Colorayme®), Diag Im- ‘mol, 19864217291 Kavanaugh A.TomarR Revel | etl. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear ant- ‘gens Arch Pathol LabMed, 200;12471-81 Tozzol R, Bizzaro N Tomut Fetal. Guidelines forthe laboratory use of autoantibody tess in the diagnosis and monitoring of autoimmune cheumatic cliseases Am] Clin Pathol 2002117:316-324 [NCCLS Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigo: (1) Indiret uorsconce Assay for Microscopy and (2) Mot ‘er Enzyme Immunoassay Methods; Approved Guidelines - Second Bcition, CLS] /LAZ-A2 200626(13), ‘Keech CL, MeChuskey J, Cordon TP Tiansfection and overexpression ofthe human 60-KDa Ro/SS-A autoantigen in HEp-2 cells. Clin. Jmmurnol mmu- ‘nopathol, 1994,73.196-151. -Kooeh CL, Howarth 8, Coates ot al. Rapid and sensitive detection of ant-Ro ($S-A) antibodies by indict immunofuoresconce of 60kDa Ro HEp-2 transfectants. Pathology. 1996285457 stzlor Mj, Miler 8) Detection of autoantibodies to $$-A/Ro by indirect immunolluorescence usinga transfected and overoxpressod human 60D Ro ‘autoantigen in HEp-2 ells, }Cin Lab Anal, 1995 9:218-228 alloc W, Tok BH. Routine imamunoflooescence detection of Ro(3S-A autoantibody using HEp-2 cells transfected with human 60 KDa RolS8-A. [Cin Pathol, 1999350:684-687 BossuytX Meus L, Mow Ae al Screening fr autoantibodies to SS-A/Ro by incre immunofluoresence using HEp-2000° cells. Ann Clin Boo- ‘chem. 200097 216-219. [Riff A, Houbruck H, Amos MD. Evaluation ofa recombinant antign onzyme-nked immunosorbent assay (ELISA) in the diagnosis of antinuclear “ntivoies (ANA) in children with cheuratc disorders. Clinical Rheumatology: May 002:21(2) 103-107 Arbuckle, MR, McCiain ML, Rubertone MV, etal: Development of autoantibodies before the clinical onset of systemic upus enjthematesus. New England Joual of Meckine 23, 340(15 1526-1533. ©Copyright 2011 Immuno Concepts NA. Lid 9825 Goethe Road, Suite 350, Sacramento, CA 95827 | 916.363.2649 | 900.251.5115,