Precautions and Cross Reactivity 1. Wait unt the reagents reach room temperature 15-25" C) before running the test. 2. In this kit the sera used forthe controls were negative forthe surface antgon of hepatic (HBsAg ) virus and for antioedies fant HCV and ant-HIV | andl with RIA technique. However iis ‘ecommended to real them as infected materials 3. Any equipment quid or ether substance that comes in rect ‘contact with human serum can be considered potentally Contaminated and hes to be inactivated after use and betore being discarded or washes, The inactivation can be cbiained by sterilizing inthe autoclave corusing sodium hypochlonte at 5 % (at leet 30 minutes) 4, Remove the material containing human serum, te material that came in contact with itor the reagents residues as hazardous wwaste, Dispose of n accordance with provisions of laws relating, Bibliography Tan E: Autosntibocies to nuctear antigens (ANA): Ther immunobiology and medicine, advances in immunology 33:167-239, 1982. Tan E, et al: The 1982 Revised crtora forthe classicaton of systemic lupus enjhematosus, Artvits and Rheumatism 25:1271-1277, 1982 Casalo SP, Friou Gi, Meyers LL: Significance of antibodies io DNA in systemic lupus erythematosus, Artits and Rheumatism 7: 379-390, ‘604. Gonzalea E, Rothfield I Inmunoglobuln class and pattem of nuclear fluorescence in systemic lupus erythematosus. The New England J. of Medicine 274:1333-1338, 1966. Biosafety in Microbiological and Biomedical Laboratories. Center for Dieesse Conta Nationa! incite of Heath, 1984 (HHS Pub. # (COC) 84-8395), Gi autoanticorpi Corrado Betterle:Piccn edtore Wishing soon, Wascioeung. Control. Kontrove. Control. Controle, Controie Solucion de tavado, Soluzione i isvagge. Soton ce lease Gove gates, Decne Cbredjios aril cml oes (coveR) se etagn cerat.Lonaes Perred abetbent paper Sausftiges Pape: a Se gas RBS PAPER] | Papel absorbente perforads. Caria assorbente cae ae ae fra Feuecateeartes pres, Tea AT ERAT, WS we a ee mmoniajeMesum mentgge, Mileu de (or) Cae DRO enage es ue evans vans ue ANA HEp2 72 est, codice 6948 ce Data di preparazione: 2016.06.08 34147 Trieste, via Flavia 122 Tel. +39 040 8997.1 Fax +39 040 280944 ‘wwrw.eurospital.com info@eurospital.com HEp2 RevS iten 2015.06.08‘diffuse type wih diffused or homogenous fuccescence of the nicleotus and cells in negative mitosis ~ PMS! 3. cumpy type with dense and homogenous fluorescence ‘grouped I masses of nucleolus and cells in positive mosis: ‘nti-fibrillarn antibodies ~ US-RNP- . Fluorescent poles in the mitotic apparatus of cells in mitosis, presence of one or two fluorescent dots on the sides of the fuceus inte cals n interphase. ratua antbodies (Of protferating cals. Recognize antigens of 100-200 KD ‘Ant cvtoplasmic antibodies (non nuclear 1: ‘Ant-mitochondtil = Filamentous granuiar Ruocescence of cytoplasm , more intense at peri-isclear level (the cytoplasm is stained ina rough way ). Anb-sbosome ‘Vary fine Muorescence of cytoplasm named ‘milky way” Ant cytoskeleton Equivalent to anti smooth muscle antibodies. The cytoplasmic or ‘oytoskeleton antibodies are stained ‘An§ Gola Apparatus Ieguiar and discontinue granular Muorescence, located ia @ cherecteraic way inthe cytoplasm aroune the nuctear membrane ‘And bsosome Characterste fuorescence with dotted granules regularly ‘isibuted in the whole cytoplasm ( weak stan inthe cytoplasm ) in the cals in interphase and in mitosis. Aas Fluorescence with fine granules concentrated in the peri-nuclesr In patients sora there may be more patterns simultaneously and some fluoroscopic patterns may be masked by others. By performing more titration is possible to highlight all the fluoroscopic patterns. Diagnostic sensitivity and speciticity \Were examined 10 sora with known patter and 10 negative sera withthe folowing results Sensitivity and specificity 100 % for all patterns ANA detectable ‘on Hep2 “The above data were obtained in comparison with the reference IFA system, ; Common reagents: Washing solution 20x common t all autoemmunty sides ‘SPECIMEN DILUENT IF! Common to ANA-AMA-ASMA-APCA and LKM code 6949, Hep? ode 6948 and nONA code 6950, do not use on Endomysium sides IgG conjugate and Evans Blue Common ‘Automatic procedure ‘Soval DAS Instrument: AP and APE ‘Specimen dition (@lso In serial dlutons) and reagents lspensaton is automatic, ‘The quantity of reagents needed is higher than the manual and ‘automaticaly calelate by te instrument. “The results on the Eurospital inetrument are similar on those obtained on manual ‘DO NOT USE THE DILUENT of specimen on EMA itis neceeeary to process simultaneously the four types of slides, use the washing solution diluted as the specimen dilvent. —- Gael Enero a wth eerent ee Specimen ron dluent Hep2 Siuent Se rouseratmouse tesues, nONA Automatic USE THE DILUTED procedure WASHING SOLUTION wth chuent FOR ALL THE SLIDES, HEp2 Revs ition 2015.08.08Manual procedure 1. Dispense 25 pi of postive control and 25 pl of negative contol on the frst sections. Dispense 25 pi of specimen ditions on the remaining sections. Make sure the quid covers each secon. Place the sles in the molsture chamber and incubate at room temperature (15:25 *C ) for 20 minutes 2. Remove the drops of specmen by iting the side on 2 piece (of absorbent paper. Avoid coss- contamination. 43.” By using a plastic squeeze botte or pipet wash the sides with a. gentle flow of washing soluton avoiding cross Contaminatons. 4. Dip the sles in a Copin jar containing the washing solution {2nd incubate for § minutes at room temperature | 16-25. C) 55. Change the washing soluton and repeat the steps 2 or 3 times. 6. By using the perforated absorbent paper dry the spaces between the sections, Make sure not to touch the sections and keep them alwaye moist 7..Add 25 lof conjugate to each section ( drag the drop with the tip to fil the whole section) 18. Place the sides in @ moisture chamber and incubste for 20 minutes at room temperature (15.25 °C) 8. Remove the drops of conjugate by titing the sides on a piace of absorbent paper 40. Repeat steps $ and 6 ‘STEP WITH EVANS BLUE OPTIONAL Evans Blue is used 5 After the conjugate Incubation . wash"twice with the washing solution. Evans Blue onthe section. In the moisture chamber and incubate for § Wash once withthe washing soluton 411. By using the perforated absorbent paper cry the spaces beiween the sections 12. Make sure not to touch the sections and keep them always: 413._ Add few drops of mounting medium and put the cover glass (onto avoid bubbles. Results Examine the slides under the fuorescence microscope using 200- 400 x magnification IF itis not possibie to read the slides on the same day of preparation, itis recommenced to keep them in the fiege and Store them in the dark il the next ay Limitation of the procedure 41. The results obtained with this test should be evaluated legen wth oer sriogcl fins and he patents males! 2. There are degree of non specite fuorescence due to heterophile antivodies or rected toward the contractile proteins, 3. Reproduotbilly of the resus epende ucon an accurate Pipetting technique, careful observation of incubation tes and temperatures and accurate washing methods. 4. Serum or conjugate residues can give a non specific background that makes the inlerpretation very diffcu. 5. Sections must be moist to avoid false postive or artefacts, 18. Use different is to dspense serum, conjugate and contol. 7._Postve and negatwe contol should be dispensed at each For best resuts make sure that you use serum not ‘contaminated from bacteria and fungus. 9." Surplus of mounting medium can lead to problems in focus the compound. 10. Overiapping of multiple antibodies pattems makes very ifficut the interpretation or hide the postvy in ‘some. auto- antbodies. Therefore it is recommended to perform serial tutions to pick out any pattern may be present HEp2 Rev5 ten 2015.06.08 Results interpretation ‘Asspecimen Is found 'o be negative if the staining ofthe nucleus is equal to orless than the negative contrel ‘There of 1:80 and 1:160 must be regarded as decision making levels that require diferent perabve behaviours. Tiere below 1:80 shouls be considered negative: “Tuers above 1:80 and below 1-160 should be considered tow positves; in the absence of epectic symptoms the rabent should fot be subjected to more detailed diagnosis but rather to monitor ‘over time! ters equal or above 1-160 should be considered ppositves and the patigts should undergo more detaled diagnosis 2st key to be suffering from an auto immune disease. We also recommend that laboratories very the consistency of these levels of decision based on the characteristics of ther ccasesipopulaion and provige information on the percentage of healthy subject or with other diseases that are positve to the above mentioned bier, Different Pattern Homogenous or cuse Fluorescence is 2 widespread inieest in the nucleus and Chromosomes of cols in mitosis. This pattem is associated to RDNA and histones in SLE Pesigheral Fomogenous fluorescence of the nucleus with reinforcement on the nuclear lamina anc cells in positive mitosis. Many sera that exhibit this pattem give a homogenous fluorescence when uted. Coarse or Fine speckled ‘The fuorescence shows fine or coarse granules scatared inthe nucleus whereas nucleolis are negative (This pattern is ‘associated to RNP, Sm, Sc70, SSB, ‘There are sitfrent morphological patterns 1. coarse and. diese granules. with nucleolus and chromosomes in negatve mites. UIRNP, U2 RNP 2. similar to patter 1 but with nuclear granules more {coarse and few in number ~ Sm, hn-RNP- 3. fing nuclear granules which are observed on slides fixed in acetone’ but disappear on compounds fied on methylated spit; cells in negative mitosis ~ SS-A 4" fine and diffuse nuclear granules with nucleolis and positive mitosis ~So70 5. fine granules, kregulary difuses with rucleolus and calls in negative mitosis Ku 16. antibodies thet recognze antigens associated with cell cycles. There is @ charactedstc change in the patiern of ‘orescence cel in diferent phases of cel cycle-PCNA. 7. few nuclear dots dotted orescence (ram 2 to 6 granules) of the nucleus of cals in interphase ; cells in negative mitosis p80 coin 3. nuclear dots — dotted Auorescence ( fom 1 to 24 franules } of nucleus that can provide a rough and weak Dackground fluorescence; cells in negative mitosss ~Sp-100 Sentomecic $32.64 fluorescent granules in cells in interphase; characteriste array of fuarescent granules in the chromesomal region in mitotic cols (CENP-A (1 7KD J: CENP-B ( B0KD J; CENP-C (140K0 ) Nucleolar ( Nuclear Dyeing the coarse particles in the nucleus ( less than 6 per nucleus ) and_can be distinguished in tis context the folowing main pattern (3 patterns) 1. speckled type with granular Muorescence of the nucleolus ‘and clis in negative mitosis, 2. RNA polymerase |, with granular fuorescence of the ruclealus and presence of few granules with dscrete and Intense Nuorescence in calls in mitosis ~ NOR-20ANA HEP2 Indirect immunofluorescence assay for the detection of class ant nuclear antigens in human serum Forin vitro diagnostic use Application field Kit for detection of cless G anf nuclear antigens in indirect immunofuorescence on epithelial cells from human laryngeal carcinoms ( Hep?) Principle ofthe method ‘The term ANA includes a family of antibodies directed against nuclear natural macromolecules native andlor denatured ‘commonly found in serum of patients with connectve ueeue diseases and systemic meumatc. ‘The benetical research of ant nuclear auto antbocies (ANA) or the presence of anticsDNA o: ani-Sm antibodies are very Important erteria. for the diagnosis of systemic Lupus cenythematosus (SLE), ‘The presence of ANA at high tter and ant Ra/SSA or ant La/SSB. antibodies isan important indicator of suspected. Sjegren ‘Syndrome, while the presence of anti Jo’ auto antibodies ie an Important Indicator of suspected dermato polymyositis: the presence of ant centromere (ACA) or enti topoisomerase (Sal70) ‘uto antibodies is an important Indicator of suspected fuse Systemic elerosie (3SSe) or imited(ISSe) and a high ter of ent RNP antibodies is an important Inlcator of suspected med connestive issue disease. ‘ANA Hep? Eurospital is dedicated to the determination in human serum of IgG antbodies directed againet the nuclear antigens on Hep2 cells fixed on the slide. The antigen-antibody complex is visualized by a fluorescence microscope Using a eecond antibody labelled with Nucrescein, ‘Slides with 2 sectons of Hepz e Postive coniral (human) TxOB mT Ready to use Negative contol human TxOe mT Ready o use 1G FITC conjugate FI (avaiable ao as @ Sagie | 1x3 mT ‘eagent cod $748 from 5.5m!) ready to use ‘Specmen ciusrt FT Txsem Ready to use Washing solution 20x Zeal Mounting Medium x6 mi Reedy to use Evans Blue Tx Toa Perforated absorbent paper 3 Gover glass, 8 Composition of supplied materialsireagents 1. Slides BE sides with 12 sections contaning Hep2 cali. ‘The sides must reach room temperature (15-25 °C) before use, 2. Positive control ‘A val containing 0.8 ml of serum ( human )in buffer solution. Ready to use 3. Negative control {A vial containing 0.8 ml of eerum (human) in buter solution, Ready to use 4. Ant! IgG conjugate A vial containing 3 ml of anti human 1gG antibodies, labelled with fluorescein isothiocyanate, in butler solution, Ready to use HEp2 RevS iten 2015.06.08 5. Specimen diluent [A val containing 58 mil of butler solution. Ready to use 5. Washing solution 20 x 2 vials containing 50 mi of concentrated buffer soliton 20x. Dilute in ested #20. 7. Mounting Medium ‘A vial containing 6 mi of glycerol in buter solution, Ready to use 8. Evans Be / {A vial containing 10 mi Ready to use 9. Perforated absorbent paper N pack of 6 10. Cover gla 1 pack of 18 Instruments and materials required but not supplied 1. Disposable pipates 20, 100p1 2. Moisture chamber 3 Copinjar for sides 4. Fluorescence microscope (with FITC fiters. Excitation 450- 1490 nm, emission 515 nm) Storage Slides and reagents must be stored at 2-8 * C and used before the expiration date printed on the label. Avoid exposure to high temperature, rect sunlight or extremely humidity condition Reagents Preparation Stability aftr first use Reagents are stable untl the expiration date printed onthe abe! when stored at2-8°C, ‘Wash buler are stable for at least 30 days when stored at 2- Stability during transportation ‘An accelerate slbilfy test proved that al reagents ae stable ater 48 hours at 37°C. Washing solution Prepare the washing solution by diluting the vial content with dstiled or deionized H2O to a fnal volume of 1000 ml If some precisitates appear in the concentrated solution, wait for the ‘Soliton to reach room temperature before dion. ‘Specimens collection ‘Sorum is required for ANA Hep2 Eurospital, It is important to preserve the integity of the blood specimen from the time of Collection upto the end of he test Perio the collecion using a non traumatic wechnique. Allow blood to dot for at least 20-30 minutes to make the clot retraction easier. Prir to centfugation remove the clot from the walls of the tubes. It is not necessary to add any preservatives to the serum specimen to maintain the integrity. Specimens must be stored at 2-8 ° C and assayed within 24 hours after collection, Tr the test cannot be pertormed within 24 hours, the serum specimen must be ttazen. Avoid repested freezing and thawing. Do not use automatic deftost freezers, because the specimens could be subjected to freezing and thawing cycles which might denature te IgG antbodies and mislead the results. ‘Avoid use of lipemic, hemolyzed, heat-treated or contaminated specmens “ Specimens dilution Specimens must be cluted before use. ‘The recommended dition is 1:80 (5 1 of specimen + 396 pl of dient) for adults and 1:40 for chidren,