MX104 - FIDIS anti-CCP -CE
Contents
1. a Remove the microplate lid verify that the vacuum break valve is closed and place it on the washer b Start the pump Stop it when the whole incubation liquid has been removed and open the vacuum break valve Close the vacuum break valve c Dispense 100uL of dilution buffer B in all the used wells d Start the pump Stop it when the whole buffer has been removed and open the vacuum break valve Close the vacuum break valve e Remove the microplate from the washer and remove the residual buffer by blotting the plate on an absorbent towel f Dispense 100uL of dilution buffer B in all the used wells Place the plate on a totally dry surface at room temperature The reading must be doing in the hour following adding the solution B During this time keep the plate at room temperature away from direct sunlight 6 Reading of the test Follow the MLX BOOSTER and FIDIS user s manual Processing batches Product Insert FIDIS anti CCP MX 104 Page 11 14 VALIDATION CRITERIA OF THE RESULTS Calibrator negative and positive controls have to be run with every batch of samples to ensure that all reagents and procedures performed properly In order to validate the results all the criteria listed below must be met Otherwise the test is invalid and must be repeated a The positive controls should show a value within the limits printed on the corresponding vial labels b The negative co2. 2 Preparation of samples and controls Dilute the samples and controls at 1 201 in dilution buffer B Ex 10uL sample in 2000uL dilution buffer B Vortex vigorously 3 Assay configuration Use the work sheet included in the kit to identify the location of the samples a When setting up the test systematically take into account the following well requirements gt See examples below 1 reagent blank well 1 well for negative control 1 well for positive control 2 calibrator wells b Calculation of the correct number of wells necessary and their location In the following examples 2 different configurations are described according to the number of wells needed and the availability of unused wells The sample dispensing must be systematically carried out in a column Do not leave empty wells The same microplate can be used for more than one serie of tests if unused wells are protected by microplate sealing film Any single well can only be used once To avoid any error in using a well more than once identify the used wells with a marker see example 2 below c Programming of the test protocol on the FIDIS Refer to the FIDIS users manual Creating a batch or a multi batch Example 1 The microplate is totally blank no well has yet been used Use A1 as the 1 well 2 3 amp n m o Le w xg Product Insert FIDIS anti CCP MX 104 Page 10 14 E
3. REACTIFS CONDITIONS D INCUBATIONS QUANTITE A DISTRIBUER Incubation des chantillons Lavage 1 Incubation du conjugu Lavage 2 Lecture D poser 50ul de microsph res dans chaque puits Tampon de dilution B blanc r actif Contr le n gatif dilu en tampon de dilution B 30 min Contr le positif dilu en tampon de dilution B Calibrateur pr t l emploi Temperature ambiante en double Echantillons dilu s en tampon de dilution B Laver 2 fois au tampon de lavage C 300ul puits 1004 Conjugu anti IgG 30 min Pr t l emploi Temp rature ambiante Laver 1 fois avec du tampon de dilution B 100ul puits Ajouter 100yl puits de tampon de dilution B et analyser par insertion de la plaque dans le cytom tre de flux En cas de lecture diff r e la plaque devra tre analys e dans l heure qui suit l ajout de la solution B Durant cette p riode la plaque doit tre conserv e temp rature ambiante et prot g e de la lumi re directe LEGENDE DES SYMBOLES A Faint 8T R 5 Risque Biologique Conserver Num ro de catalogue 2 Lire le manuel d utilisation Far por LOT Num ro de lot Nombre de tests bg A utiliser avant C Communaut Europ enne Microplaque contRoL Contr le n gatif Microspheres Diluant chantillon CONTROL Contr le positif Conjugu IgG MICROSPHERES Tampon de reconstitution des Tampon de lavage microsp
4. eo j P contact nes subsidiaries or distributors 1 Start the run as described in the Users Manual NOTE The FIDIS takes 30 minutes to warm up after being turned on A new warm up is necessary after 4 hours of system inactivity 2 Calibration and controls are described in the User s Manual Calibration and controls should be routinely performed once per month and each time a new lot of Sheath fluid reagent is used in order to insure optimal instrument performance Calibration should also be performed when the temperature is out of the determined range shown on the run batch screen 3 Programming a batch or a multibatch of test protocol on the FIDIS as described in the User s Manual 4 Load the microplate into the plate holder of the FIDIS as described in the User s Manual 5 Analyze the results according the User s Manual 6 When finished for the day perform the sanitizing and soak operations prior to turning the Analyzer off according to the shutting down procedure described in the User s Manual Reagents Preparation Remove the individual components from storage allow them to warm up to room temperature 18 C to 25 C and mix them well 1 Microsphere preparation Reconstitute the microsphere solution by adding the vial D of reconstitution buffer to the vial A of lyophilized microspheres Wait 5 minutes and vortex Stable 2 months at 2 C to 8 after reconstitution
5. 14 CRITERES DE VALIDATION DES RESULTATS Le calibrateur les contr les positif et n gatif doivent tre test s dans chaque s rie d essai pour s assurer que tous les r actifs et proc dures ont t ex cut s correctement Afin de valider les r sultats tous les crit res num r s ci dessous doivent tre rencontr s En cas de non conformit d un de ces crit res le test devra tre consid r comme non valide et l analyse devra tre refaite a La valeur du contr le positif doit tre comprise dans les limites de celles indiqu es sur les tiquettes des flacons correspondants b La valeur du contr le n gatif doit tre inf rieure 25 UA ml La solution de microsph res contient des billes t moins permettant de v rifier la pr sence de s rum dans les puits ainsi que le bon d roulement du test Un puits pr sentant un signal r ponse non conforme sera invalid par le logiciel MLX BOOSTERTM INTERPRETATION DES RESULTATS CALCUL DES RESULTATS Les r sultats sont automatiquement calcul s par le logiciel MLX BOOSTERTM et peuvent tre imprim s pour chaque analyse LIMITES Les s rums h molys s lip miques ict riques pr sentant des taux lev s en IgG monoclonales des complexes immuns ou des facteurs rhumato des peuvent entra ner des r sultats faussement positifs Les s rums de patients immunod prim s donneront un r sultat non valide CONTROL
6. anti CCP antibodies for example ASSAY PRINCIPLE Rheumatoid arthritis is one of the most widespread systemic auto immune diseases The prevalence of the disease reaches 0 5 to 1 of adult population The diagnostic of rneumathoid arthritis is essentially based on clinical radiological and immunological characteristics The measurement of Rheumatoid Factor RF is widely used Althought RF test is sensitive the specificity is low Indeed RF are found in patients with rheumatoid diseases inflammatory or auto immune diseases as well as patients subject to chronic infections and in healthy population In 1998 Schellekens et al showed that auto antibodies directed against synthetic citrullinated peptides were very specific for rheumatoid arthritis Further studies shown that auto antibodies directed against cyclic citrullinated peptides were specific for rheumatoid arthritis and were more sensitive than linear peptide It has been shown that anti CCP antibodies can be detected at the onset of the disease before any kind of clinical symptoms Several publications show that a high level of anti CCP antibodies is correlated to a very active disease These results demonstrate the importance of the anti CCP antibodies for the diagnosis of rheumatoid arthritis in the early state of the disease FIDIS anti CCP is based on the use of distinct uniform size color coded microspheres and a benchtop flow cytometer inte
7. associated with systemic autoimmune diseases using the Luminex technology Results of a multicenter study Ann Biol Clin Paris 2005 Jan Feb 63 1 51 8 Rouquette A M Desgruelles C Laroche P Evaluation of the new multiplexed immunoassay FIDIS for simultaneous quantitative determination of antinuclear antibodies and comparison with conventional methods Hematology Laboratory Tenon Hospital 75020 Paris France Am J Clin Pathol 2003 120 5 676 81 Product Insert FIDIS anti CCP MX 104 Page 13 14 SUMMARY OF THE TEST PROCEDURE INCUBATION VOLUME TO BE DISTRIBUTED REAGENT CE o S v GENTS CONDITIONS Dispense 50uL of microspheres reagent in each well to be used Dilution buffer B reagent blank Diluted negative control in dilution buffer B Incubation Diluted positive control 30 min of the samples in dilution buffer B Room temperature Ready to use Calibrator in duplicate Diluted samples in dilution buffer B Washing 1 Wash twice in washing buffer C 300uL well Incubation 30 min cu wo Sec of the conjugate nu IgG conjugate Room temperature Washing 2 Wash 1 time in Dilution buffer B 100uL well Add 100uL well of Dilution buffer B and proceed immediately with the reading in FIDIS Readin Instrument g The reading must be doing in the hour following adding the solution B During this time keep the plate at room temperature away from direct sunlight SYMBOL
8. blanc DIL SPE 2x115ml Pr t l emploi Flacon de calibrateur CAL Pr t l emploi 1x1 5ml Les titres sont indiqu s sur l tiquette du flacon Flacon de contr le positif donnant une r activit standardis e et constituant un contr le de r action destin v rifier l activit des r actifs et le bon fonctionnement de l essai 1x2 A diluer x 250 Les valeurs attendues sont indiqu es sur l tiquette du flacon Flacon de contr le n gatif A diluer CONTROL 1x 250ul Flacon de conjugu anti lgG humaine coupl a la phyco rythrine CONJ IgG 1x 12ml Pr t a emploi Flacon C de tampon de lavage flacon noir Pr t a emploi WASH 1 x 100ml Flacon D de tampon de reconstitution des microsph res 1 MICROSPHERES xem Pr t l emploi Les titres du calibrateur sont exprim s en unit s arbitraires par ml UA ml Notice technique FIDIS anti CCP MX 104 Page 2 14 MATERIEL NECESSAIRE NON FOURNI Pipettes de pr cision capables de d livrer pr cis ment de 5ul a 1000pl Pipette multicanaux ou distributeur r p titif capables de d livrer pr cis ment de 40ul 300ul ou 5yil a 2ml Agitateur Chronom tre Sonicateur Papier absorbant Pipettes s rologiques Films pour microplaques ECHANTILLONS Utiliser du s rum Eviter d utiliser des s rums lip miques ou h molys s ainsi que des pr l vements congel s
9. e The reaction is then directly measured by the flow cytometer which differentiates each set of microspheres according to its fluorescence color while simultaneously measuring the average fluorescence emitted by the conjugate e A calibration system allows the determination of the titer AU mL of each sample by interpolation for each following antigenic specificity HCP1 HCP2 VCP1 and VCP2 The FIDIS anti CCP kit could be used with CARIS system diluting and dispensing device CAUTION SPECIMEN COLLECTION AND HANDLING The test is performed on serum Lipemic sera should be avoided as well as samples which have been frozen and defrosted more than once If the test is not run immediately the samples should be stored at 2 C 8 C for a maximum of 5 days for longer storage frozen undiluted samples at 20 C To avoid non specific binding it is recommended to centrifuge and filter the cloudy samples or samples frozen for more than 6 months REAGENTS SUPPLIED 96 wells microplate with filtering membrane and lid 1 plate Vial A of color coded microsphere set of 4 sensitized by HCP1 HCP2 VCP1 and VCP2 synthetic peptides Lyophilized to be reconstituted with the buffer named qs 6mL D MICROSPHERES Reagents in solution contain less of 0 1 w v sodium azide and 0 02 w v of Proclin 300 Do not eat and avoid contact with skin and eyes Azide can form explosive mi
10. et d congel s plus d une fois Si le dosage n est pas effectu imm diatement les chantillons devront tre conserv s r frig r s entre 2 C et 8 C pendant 5 jours maximum Au del ils devront tre congel s a 20 C Afin de limiter toute fixation non sp cifique il est conseill de centrifuger et de filtrer les chantillons congel s depuis plus de 6 mois et troubles PREPARATION DU TEST Pr paration de l unit de filtration FIDIS V rifier les tuyaux reliant la pompe au support et le r glage du manom tre molette totalement ferm e Utilisation du syst me d analyse FIDIS et du logiciel MLX BOOSTER Se reporter au Manuel d utilisation fourni avec le syst me FIDIS pour effectuer les tapes de mise en route et de calcul Pour toutes informations compl mentaires et ou r solution de probl mes veuillez x ee prendre contact aupr s de Teri ou de votre distributeur 1 Commencer la s rie comme indiqu e dans le Manuel d utilisation N B L appareil met 30 minutes pour chauffer apr s avoir t allum Un nouveau pr chauffage est n cessaire apr s 4 heures d inactivit du syst me 2 Les tapes de calibration et de contr les sont d crites dans le Manuel d utilisation Ces 2 tapes devront tre ex cut es r guli rement 1 fois mois et chaque nouveau lot de Sheath fluid afin d assurer une performance optimale de l appareil L tape de calibration d
11. et supprimer rapidement le vide en ouvrant le robinet casse vide Refermer le robinet casse vide e Distribuer 3800 de tampon de lavage C f D clencher la pompe Apr s aspiration totale du liquide compter 5 secondes suppl mentaires arr ter la pompe et supprimer rapidement le vide en ouvrant le robinet casse vide Refermer le robinet casse vide g Retirer la microplaque du laveur et liminer le tampon r siduel en tapant fortement sa base 10 fois sur du papier absorbant h Repositionner la plaque sur le laveur et filtrer nouveau 5 secondes Arr ter la pompe et supprimer rapidement le vide en ouvrant le robinet casse vide i Retirer la microplaque du laveur et liminer le tampon r siduel en tapant fortement sa base sur un papier absorbant j Placer ensuite la microplaque sur une surface totalement s che avant de distribuer le conjugu 4 Incubation du conjugu D poser 100ul de conjugu Laisser incuber 30 minutes temp rature ambiante sans agitation en recouvrant la plaque et en vitant de la placer sous la lumi re directe Le temps d incubation d bute apr s que le conjugu ait t ajout tous les puits Si ce temps n est pas respect les r sultats pourront tre erron s Remarque le conjugu est sensible la lumi re gt Refermer le flacon apr s utilisation 5 Lavage 2 Laver la plaque en utilisant unit de filtration par 1 cycle en
12. par filtration permet d liminer les l ments non fix s e Un anticorps secondaire conjugu la phyco rythrine dirig contre les immunoglobulines humaines d isotype IgG permet de r v ler les anticorps pr c demment captur s e Une tape finale de lavage stoppe la r action et permet d liminer les anticorps non li s e La r action est alors mesur e par le cytom tre en flux qui identifie chaque type de microspheres et mesure la fluorescence moyenne du conjugu fix e Un syst me de calibration permet par interpolation de d finir la valeur de l chantillon en unit arbitraire UA ml sur chacune des sp cificit s antig niques suivantes HCP1 HCP2 VCP1 et VCP2 Le coffret FIDIS anti CCP 9 peut tre utilis avec l automate de dilution r partition CARIS PRECAUTIONS D EMPLOI STABILITE ET CONDITIONS DE CONSERVATION Tous les r actifs doivent tre conserv s entre 2 C et 8 C dans leur conditionnement d origine Ne pas congeler les r actifs Ne pas utiliser un coffret dont les dates de p remption sont d pass es Remettre imm diatement entre 2 C et 8 les r actifs non utilis s COMPOSITION DU COFFRET Les r actifs en solutions contiennent comme agent de conservation de l azide de sodium une concentration lt 0 1 w v ou du ProClin 300 0 02 w v Ne pas avaler et viter tout contact avec la peau et les muqueuses L a
13. sultat de chaque sp cificit est inf rieur 25 UA ml c Un chantillon est consid r limite lorsqu au moins une des sp cificit s pr sente un titre en anticorps compris entre 25 et 30 UA ml Notice technique FIDIS anti CCP MX 104 Page 5 14 BIBLIOGRAPHIE WO2004087747 2004 10 14 Citrullinated synthetic peptides and uses thereof Inv Migliorini Paola WO 2011 061720 2011 05 26 Viral citrullinated peptides and uses thereof Inv PRATESI Federico MIGLIORINI Paola WO 2012 001103 2012 01 05 Histone citrullinated peptides and uses thereof Inv PRATESI Federico ALCARO Maria Claudia CHELLI Mario LOLLI Francesco PAOLINI Ilaria PAPINI Anna Maria ROVERO Paolo MIGLIORINI Paola Pratesi F Tommasi C Anzilotti C Puxeddu I Sardano E Di Colo G Migliorini P Antibodies to a new viral citrullinated peptide VCP2 fine specificity and correlation with anti cyclic citrullinated peptide CCP and anti VCP1 antibodies Clin Exp Immunol 2011 164 337 Anzilotti C Merlini G Pratesi F Tommasi C Chimenti D Migliorini P Antibodies to viral citrullinated peptide in rheumatoid arthritis J Rheumatol 2006 Apr 33 4 647 51 Arnett FC Edworthy SM Bloch DA et al The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis Arthritis Rheum 1988 31 3 315 24 Van Venrooij WJ Hazes JM Visser H Anticitrullinate
14. tampon de dilution B Utiliser de pr f rence deux r servoirs r actifs distincts pour les lavages 1 et 2 Ou bien nettoyer et s cher correctement le r servoir entre les deux lavages afin de ne pas m langer les deux tampons B et C i a Retirer le couvercle de la microplaque et la positionner sur l unit de filtration v rifier que le robinet casse vide est en position ferm e b D clencher la pompe D s la disparition totale du liquide arr ter la pompe et supprimer rapidement le vide par ouverture du robinet casse vide Refermer le robinet casse vide c Distribuer 100ul de tampon de dilution B d D clencher la pompe D s la disparition totale du liquide arr ter la pompe et supprimer rapidement le vide en ouvrant le robinet casse vide Refermer le robinet casse vide e Retirer la microplaque du laveur et liminer le tampon r siduel en tapant fortement sa base sur un papier absorbant f Distribuer 100ul de tampon de dilution B et proc der l analyse de la microplaque En cas de lecture diff r e la plaque devra tre analys e dans l heure qui suit l ajout de la solution B Durant cette p riode la plaque doit tre conserv e a temp rature ambiante et prot g e de la lumi re directe 6 Analyse L effectuer conform ment au manuel d utilisation FIDIS et MLX BOOSTER Notice technique FIDIS anti CCP MX 104 Page 4
15. Be Theradiag FIDIS anti CCP MX 104 DEFINITION Le coffret FIDIS anti CCP e49 constitue une m thode d identification semi quantitative d autoanticorps sur supports particulaires utilisant un syst me de d tection par cytom trie de flux Il permet la recherche simultan e de 4 anticorps d isotype IgG dirig s contre 4 peptides citrullin s HCP1 HCP2 VCP1 et VCP2 Ces 4 peptides synth tiques sont issus de prot ines humaines HCP1 et HCP2 et virales VCP1 et VCP2 Les anticorps d tect s appartiennent a la famille des anticorps anti peptides citrullin s ACPA et sont capables de d tecter les peptides cycliques citrullin s CCP ainsi que le fibrinog ne d imin VALEUR DIAGNOSTIQUE Fr AVRIL 2013 D om S oO S En 2008 la recherche des anticorps anti CCP a t introduite a la Nomenclature des Actes de Biologie M dicale en France En 2010 American College of Rheumatology ACR et European League Against Rheumatism EULAR ont publi conjointement de nouveaux crit res de classification pour la polyarthrite rhumato de Pour la premi re fois la mesure des ACPA Anti Citrullinated Peptides Antibodies tels que les anti CCP a t int gr e ces crit res PRINCIPE DU TEST La polyarthrite rhumato de PR est l une des maladies auto immunes syst miques la plus r pandue au monde Sa pr valence atteint 0 5 1 0 de la population adulte Le diagno
16. E DE QUALITE Il est conseill d utiliser des contr les internes ou externes pour les diff rentes sp cificit s FIDELITE DU TEST Intra essai Inter essais 30 tests dans le m me 2 tests dans 20 essais gt i Pme ae essai diff rents D terminer pour chacune des sp cificit s la ASE VAR i eae z cificit 5 N pr sence ou l absence d anticorps selon le tableau aniloenide mereen CV Peya CV suivant HCP1 48 6 5 55 9 Unit s arbitraires UA mI lt 25 UA mI 25 30 UA mI gt 30 UA mI HCP2 37 8 5 40 8 VCP1 36 6 5 40 9 Interpr tation N gatif Limite Positif VCP2 42 7 41 7 Les r sultats limites doivent tre contr l s sur un second pr l vement et interpr t s en fonction d examens compl mentaires et du contexte clinique Chaque laboratoire doit tablir et conserver ses propres valeurs de plage normale de r f rence en fonction de la population de patients et d autres facteurs locaux D terminer le statut final de l chantillon a Un chantillon est consid r comme positif lorsqu au moins un des r sultats obtenus pour les sp cificit s HCP1 HCP2 VCP1 et VCP2 est trouv positif En d autres termes un r sultat est consid r comme positif si au moins une des sp cificit s pr sentes a un titre en anticorps sup rieur a 30 UA ml b Un chantillon est consid r n gatif lorsque le r
17. S USED P A 8T Biological risk die Temperature limitation REF Catalog Number AT P REF g Read instructions for use In Vitro Diagnostic Use LOT Lot Number Number of tests Use by C EC Declaration of Conformity ay Microplate CONTROL Negative control MICROSPH ERES Microspheres DIL SPE Specimen diluent CONTROL Positive control CONJ IgG IgG Conjugate CAL Calibrator BUF IWASH Wash Buffer eo Ce oO FIDIS test Manufacturer eo Microsphere reconstitution MICROSPHERES a s Theradiag C 4 6 bld de Beaubourg CS 90138 CROISSY BEAUBOURG Tel 33 0 1 64 62 10 12 E mail support theradiag com 77435 MARNE LA VALLEE CX2 Fax 33 0 1 64 62 09 66 Internet www theradiag com France M Product Insert FIDIS anti CCP MX 104 Page 14 14
18. aille uniforme diff remment color es et d un cytom tre en flux interfac avec un syst me informatique de digitalisation et de traitement du signal Une diode rouge du cytom tre en flux en classant chaque cat gorie de microsph res sur la base de sa fluorescence unique du rouge lorange permet l identification du param tre analys Parall lement un laser vert excite la fluorescence d un compos secondaire pour quantifier la r action sp cifique qui y est associ e Chaque antig ne n cessaire la r alisation du test peptides synth tiques HCP1 HCP2 VCP1 et VCP2 est coupl une cat gorie de microsph res color es Les diff rentes cat gories sont ensuite m lang es pour constituer le r actif final support des r actions immunologiques sp cifiques avec les diff rents autoanticorps recherch s Le coffret FIDIS anti CCP lt 3 permet de d tecter 4 anticorps sp cifiques les anticorps anti HCP1 anti HCP2 anti VCP1 et anti VCP2 Le test est r alis dans une microplaque de filtration de 96 puits Notice technique FIDIS M anti CCP MX 104 Page 1 14 e Au cours d une premi re tape les chantillons dilu s des patients a tester sont incub s en pr sence des microsph res Si l chantillon contient un ou plusieurs anticorps recherch s ceux ci vont se fixer au x antig ne s correspondant s sur les diff rentes cat gories de microsph res e Apr s incubation un lavage
19. d CONTROL 1 x 250uL Vial of anti human IgG coupled to phycoerythrin Ready eady to use CONJ IgG 1x12mL Vial C of washing buffer black vial Ready to use BUF 1x 100mL H o I Vial D of reconstitution buffer for the microsphere set 1 x 6mL MICROSPHERES Ready to use Calibrator titers are expressed in arbitrary units per ml AU ml Product Insert FIDIS anti CCP MX 104 Page 9 14 ADDITIONAL MATERIAL NOT SUPPLIED Precision pipettes capable of accurately delivering SuL to 1000uL Multichannel Pipettes or dispensers capable of accurately delivering 40uL to 300uL or 5uL to 2mL Vortex mixer Laboratory timer to monitor incubation steps Ultrasonic bath Absorbent towel Serological pipettes Microplate sealing films STABILITY AND STORAGE Store reagents in their original packaging at 2 C to 8 C Do not freeze reagents Do not use kits beyond the expiration date After use store all components immediately back at 2 C to 8 C TEST SET UP FIDIS Washer Check the tubing connecting the pump to the stand and the manometer setting wheel completely closed Using FIDIS Analyzer and MLX BOOSTER Software See the Users Manual provided with the FIDIS Instrument for detailed instructions on running the equipment and for calculation For additional information and or trouble shooting problems please
20. d protein peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis Neth J Med 2002 60 10 383 8 Nienhuis RL Mandema E Smids C A new serum factor in patients with rheumatoid arthritis The antiperinuclear factor Ann Rheum Dis 1964 23 302 05 Young BJ Mallya RK Leslie RD et al Anti keratin antibodies in rheumatoid arthritis Br Med J 1979 2 97 9 Hoet RM Boerbooms AM Arends M et al Antiperinuclear factor a marker autoantibody for rheumatoid arthritis colocalisation of the perinuclear factor and profilaggrin Ann Rheum Dis 1991 50 611 8 Sebbag M Simon M Vincent C et al The antiperinuclear factor and the so called antikeratin antibodies are the same rheumatoid arthritis specific autoantibodies J Clin Invest 1995 95 2672 9 Schellekens GA de Jong BA van den Hoogen FH et al Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis specific autoantibodies J Clin Invest 1998 101 1 273 81 Vossenaar ER van Venrooij WJ Anti CCP antibodies a highly specific marker for early rheumatoid arthritis Clin Applied Immunol Rev 2004 4 239 62 Pruijn GJ Vossenaar ER Drijfhout JW et al Anti CCP antibody detection facilitates early diagnosis and prognosis of rheumatoid arthritis Current Rheumatology Reviews 2005 1 1 1 7 Rantapaa Dahlqvist S de Jong BA Berglin E et al Antibodies against cyclic citullinated peptide and IgA rheumatoid facto
21. genic determinants recognized by rheumatoid arthritis specific autoantibodies J Clin Invest 1998 101 1 273 81 Vossenaar ER van Venrooij WJ Anti CCP antibodies a highly specific marker for early rheumatoid arthritis Clin Applied Immunol Rev 2004 4 239 62 Pruijn GJ Vossenaar ER Drijfhout JW et al Anti CCP antibody detection facilitates early diagnosis and prognosis of rheumatoid arthritis Current Rheumatology Reviews 2005 1 1 1 7 Rantapaa Dahlqvist S de Jong BA Berglin E et al Antibodies against cyclic citullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis Arthirtis Rheum 2003 48 10 2741 49 Meyer O Labarre C Dougados M et al Anticitrullinated protein peptide antibody assays in early Rheumatoid Arthritis for predicting five year radiographic damage Ann Rheum Dis 2003 62 120 26 Aletaha D Neogi T Silman AJ et al Rheumatoid Arthritis Classification Ciriteria An American College of Rheumatology European League against Rheumatism Collaborative Initiative Arthritis Rheum 2010 62 9 2569 81 Susan S Copple Thomas B Martins C Masterson E Joly Harry R Hill Comparison of Three Multiplex Immunoassays for Detection of Antibodies to Extractable Nuclear Antigen Using Clinically Defined Sera Ann N Y Acad Sci 1109 464 472 2007 Buliard A Fortenfant F Ghillani Dalbin P Musset L Oksman F Olsson N O Related Articles Links Analysis of nine autoantibodies
22. h res Calibrateur Test FIDIS Fabricant al z E gt z a Theradeg VD CE 4 6 bld de Beaubourg CS 90138 CROISSY BEAUBOURG T l 01 64 62 10 12 E mail support theradiag com 77435 MARNE LA VALLEE CX2 Fax 01 64 62 09 66 Internet www theradiag com France Notice technique FIDIS anti CCP MX 104 Page 7 14 s Theradiag FIDIS anti CCP MX 104 INTENDED USE FIDIS anti CCP e is a semi quantitative homogeneous fluorescent based microparticles immunoassay using flow cytometery readings It is designed for the simultaneous detection of 4 autoantibodies IgG isotype directed against 4 synthetic citrullinated peptides HCP1 HCP2 VCP1 and VCP2 HCP1 and HCP2 are from human protein origin whereas VCP1 and VCP2 are from viral protein origin These autoantibodies are able to detect cyclic citrullinated peptides CCP and deiminated fibrinogen and are associated to anti citrullinated peptide protein antibodies ACPA SUMMARY AND DIAGNOSTIC VALUES Ag APRIL 2013 In 2008 the search for anti CCP antibodies was introduced to the Nomenclature of the Medical Biology s acts in France In 2010 the American College of Rheumatology ACR and the European League Against Rheumatism EULAR published criteria for the classification of rheumatoid arthritis For the first time serological criteria included the measurement of anti citrullinated peptide protein antibodies ACPA such as
23. inated peptide VCP2 fine specificity and correlation with anti cyclic citrullinated peptide CCP and anti VCP1 antibodies Clin Exp Immunol 2011 164 337 Anzilotti C Merlini G Pratesi F Tommasi C Chimenti D Migliorini P Antibodies to viral citrullinated peptide in rheumatoid arthritis J Rheumatol 2006 Apr 33 4 647 51 Arnett FC Edworthy SM Bloch DA et al The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis Arthritis Rheum 1988 31 3 315 24 Van Venrooij WJ Hazes JM Visser H Anticitrullinated protein peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis Neth J Med 2002 60 10 383 8 Nienhuis RL Mandema E Smids C A new serum factor in patients with rheumatoid arthritis The antiperinuclear factor Ann Rheum Dis 1964 23 302 05 Young BJ Mallya RK Leslie RD et al Anti keratin antibodies in rheumatoid arthritis Br Med J 1979 2 97 9 Hoet RM Boerbooms AM Arends M et al Antiperinuclear factor a marker autoantibody for rheumatoid arthritis colocalisation of the perinuclear factor and profilaggrin Ann Rheum Dis 1991 50 611 8 Sebbag M Simon M Vincent C et al The antiperinuclear factor and the so called antikeratin antibodies are the same rheumatoid arthritis specific autoantibodies J Clin Invest 1995 95 2672 9 Schellekens GA de Jong BA van den Hoogen FH et al Citrulline is an essential constituent of anti
24. non utilis s avec des films plastiques si n cessaire D poser 50ul de microsph res dans chaque puits apr s avoir pr alablement agit le flacon vigoureusement au vortex pendant 20s REMARQUE Dans le cadre de l utilisation de l automate CARIS une sonication des microsph res de 5 minutes est pr conis e Dans le cadre de l utilisation d un automate de dilution r partition les tapes 1 et 2 doivent tre invers es gt distribuer le s rum dilu puis les microspheres 2 Incubation des chantillons D poser 100u de tampon de dilution B pour le blanc r actif D poser 100ul de contr les dilu s de calibrateur pr t l emploi et d chantillons dilu s Laisser incuber 30 minutes temp rature ambiante sans agitation en recouvrant la plaque et en vitant de la laisser sous la lumi re directe 3 Lavage 1 Laver la plaque en utilisant l unit de filtration par 2 cycles successifs en tampon de lavage C a Retirer le couvercle de la microplaque et la positionner sur l unit de filtration v rifier que le robinet casse vide est en position ferm e b D clencher la pompe D s la disparition totale du liquide arr ter la pompe et supprimer rapidement le vide en ouvrant le robinet casse vide Refermer le robinet casse vide c Distribuer 800 de tampon de lavage C d D clencher la pompe D s la disparition totale du liquide arr ter la pompe
25. noter la localisation des chantillons a Pr voir syst matiquement 4 1 puits blanc r actif e 1 puits pour le contr le n gatif 7 1 puits pour le contr le positif 2 puits calibrateur b D termination du nombre exact de puits n cessaires et de leur attribution Les sch mas qui suivent donnent 2 exemples de configuration selon le nombre de puits n cessaires et la disponibilit en puits vierges Le sens du d p t doit s effectuer syst matiquement par colonne sans intercaler de puits vides Une m me plaque peut tre utilis e au cours de diff rents essais si les puits non utilis s sont prot g s par des films pour microplaque Un m me puits ne peut tre r utilis plusieurs fois Afin d viter toute erreur dans ce sens il est conseill d identifier les puits d j usag s voir exemple 2 ci dessous c Programmation du protocole d analyses au niveau du FIDIS Se reporter au manuel d utilisation FIDIS Programmation d un batch ou d un Multibatch Exemple 1 la plaque est totalement vierge l attribution des puits d bute en A1 ce I 2 OS 00e D en Cer Cs Ex BIO gO OS OOO OOo I 0 Notice technique FIDIS anti CCP MX 104 Page 3 14 Exemple 2 16 puits ont d j t utilis s l attribution des puits d bute en 1 2 9 3 4 5 6 7 8 10 11 12 MODE OPERATOIRE 1 Distribution des microsph res Prot ger les puits
26. ntrols should be less than 25 AU mL The microsphere set contains Internal standard beads allowing to verify the presence of serum in wells as well as the good respect of the test procedure Wells presenting a not corresponding fluorescent signal will be no valid with MLX BOOSTER software INTERPRETATION OF RESULTS Determine for each specificity the presence or the absence of antibodies according to the following table Arbitrary Units AU mL lt 25 AU mL 25 30 AU mL gt 30 AU mL Interpretation Negative Equivocal Positive Borderline results should be controlled on a second sample and the interpretation of the results should be done in the frame of additional testing and taking into account the clinical status of the patient Each laboratory should establish and maintain its own references normal range values based on the patient population and other local factors Determine the final status of the sample a A sample is considered positive when at least one result from HCP1 HCP2 VCP1 and VCP2 antibody specificity is found positive In other terms a result is considered positive if at least one result from each antibody specificity is higher than 30 AU mL b A sample is considered negative when the result from each specificity is less than 25 AU mL c A sample is considered equivocal when at least one of the result is compri
27. oit galement tre effectu e si la temp rature indiqu e sur l cran de la s rie du lot est en dehors de celle param tr e 3 Programmer un lot ou une s rie de lots multiples sur FIDIS comme indiqu dans le Manuel d utilisation 4 Placer la microplaque dans le support de plaque du syst me FIDIS comme indiqu dans le Manuel d utilisation 5 Effectuer l analyse des r sultats comme indiqu dans le Manuel d utilisation 6 A la fin de la derni re utilisation journali re de l appareil ex cuter les op rations de lavage et de d sinfection avant d arr ter le syst me d analyse conform ment aux instructions d arr t d crites dans le Manuel d utilisation Pr paration des r actifs Ramener l ensemble des r actifs temp rature ambiante 18 C 25 C avant de les pr parer extemporan ment 1 Pr paration des microsph res Reconstituer la solution de microsph re en ajoutant tout le flacon D de tampon de reconstitution des billes au flacon A Attendre 5 minutes et vortexer Dur e de conservation apr s reprise 2 mois entre 2 et 8 C 2 Pr paration des chantillons et des contr les Diluer les chantillons et les contr les au 1 201 dans le tampon de dilution B Exemple 1Oul d chantillon dans 2000u de tampon de dilution B Agjiter vigoureusement au vortex 3 D finition de la configuration du test Utiliser la feuille de travail contenue dans le coffret pour
28. r predict the development of rheumatoid arthritis Arthirtis Rheum 2003 48 10 2741 49 Meyer O Labarre C Dougados M et al Anticitrullinated protein peptide antibody assays in early Rheumatoid Arthritis for predicting five year radiographic damage Ann Rheum Dis 2003 62 120 26 Aletaha D Neogi T Silman AJ et al Rheumatoid Arthritis Classification Ciriteria An American College of Rheumatology European League against Rheumatism Collaborative Initiative Arthritis Rheum 2010 62 9 2569 81 Susan S Copple Thomas B Martins C Masterson E Joly Harry R Hill Comparison of Three Multiplex Immunoassays for Detection of Antibodies to Extractable Nuclear Antigen Using Clinically Defined Sera Ann N Y Acad Sci 1109 464 472 2007 Buliard A Fortenfant F Ghillani Dalbin P Musset L Oksman F Olsson N O Related Articles Links Analysis of nine autoantibodies associated with systemic autoimmune diseases using the Luminex technology Results of a multicenter study Ann Biol Clin Paris 2005 Jan Feb 63 1 51 8 Rouquette A M Desgruelles C Laroche P Evaluation of the new multiplexed immunoassay FIDIS for simultaneous quantitative determination of antinuclear antibodies and comparison with conventional methods Hematology Laboratory Tenon Hospital 75020 Paris France Am J Clin Pathol 2003 120 5 676 81 Notice technique FIDIS anti CCP MX 104 Page 6 14 RECAPITULATIF DU MODE OPERATOIRE
29. reak valve Close the vacuum break valve Dispense 300uL of washing buffer C in all the used wells Start the pump Stop it after 5 additional seconds when all the buffer has been removed and open the vacuum break valve Close the vacuum break valve g Remove the microplate from the washer and remove the residual buffer by tapping vigorously the plate 10 times on an absorbent towel h Place again the microplate on the washer and start the pump for 5 seconds Stop it and quickly open the vacuum break valve i Remove the microplate from the washer and remove the residual buffer by blotting vigorously the plate on an absorbent towel j Place the plate on a totally dry surface before starting the conjugate incubation 4 Incubation of the conjugate Dispense 100uL of ready to use conjugate in each well used Cover the plate and incubate 30 minutes at room temperature away from direct sunlight and without shaking The incubation time starts after the conjugate has been added to all wells If this timing is not followed the results might be erroneous Note the conjugate is photosensitive gt After using close the vial carefully 5 Wash step 2 Wash the plate using the filtration unit on 1 cycle with dilution buffer B Use two different reagent reservoirs for the 2 washing steps Either clean and dry correctly the reservoir between both wash steps avoid to mix both B and C buffers
30. rfaced to digital signal processing hardware and software A red diode laser beam in the flow cytometer classifies each set of microspheres on the basis of its unique fluorescence intensity red to orange thus identifying which analyte is being tested At the same time a green laser beam illuminates the external second molecule fluorescence to quantify the reaction related to the specific analyte Each antigen synthetic peptides HCP1 HCP2 VCP1 and VCP2 required for the assay is coupled to an individual set of microspheres through its surface functional groups The different peptides coupled microspheres are mixed together constituting the final microsphere reagent The FIDIS anti CCP 9 allows the detection of 4 autoantibody specificities anti HCP1 anti HCP2 anti VCP1 and anti VCP2 Product Insert FIDIS anti CCP MX 104 Page 8 14 The test is performed in a 96 well microplate with a filtering membrane e In the first step the sample is distributed in each well containing the microspheres mixture If this sample contains one or more of the suspected antibodies this ese antibody ies bind to the corresponding antigen s on the various sets of microspheres e After incubation a wash step using a filtration process removes the unbound antibodies e A phycoerythrin labelled anti human IgG conjugate is then added that binds to the previously bound antibodies e A final wash step allows to stop the reaction
31. sed between 25 AU mL and 30 AU mL CALCUL OF RESULTS The results are automatically calculated by MLX BOOSTER software and can be printed for each assay LIMITATION Hemolytic lipemic icteric samples or samples with abnormal concentration of IgG and or complement levels or samples with rheumatoid factor may confound the results of this assay The serums from Immunodeficient patients will give a no valid result QUALITY CONTROL It is recommended to use internally and externally sourced control material for the different specificities REPRODUCIBILITY Within run Between run a se inthe same run 2 isik 20 different runs spe E ee Od E HCP1 48 6 5 55 9 HCP2 37 8 5 40 8 VCP1 36 6 5 40 9 VCP2 42 7 41 7 Product Insert FIDIS anti CCP MX 104 Page 12 14 BIBLIOGRAPHY WO2004087747 2004 10 14 Citrullinated synthetic peptides and uses thereof Inv Migliorini Paola WO 2011 061720 2011 05 26 Viral citrullinated peptides and uses thereof Inv PRATESI Federico MIGLIORINI Paola WO 2012 001103 2012 01 05 Histone citrullinated peptides and uses thereof Inv PRATESI Federico ALCARO Maria Claudia CHELLI Mario LOLLI Francesco PAOLINI Ilaria PAPINI Anna Maria ROVERO Paolo MIGLIORINI Paola Pratesi F Tommasi C Anzilotti C Puxeddu I Sardano E Di Colo G Migliorini P Antibodies to a new viral citrull
32. stic de la polyarthrite rhumato de repose essentiellement sur des caract ristiques cliniques radiologiques et immunologiques Le test s rologique le plus commun est la mesure du facteur rhumato de FR dans le s rum Bien que le test du FR pr sente une bonne sensibilit ce param tre manque de sp cificit Les FR sont galement retrouv s chez des patients atteints d autres maladies rhumatismales inflammatoires ou auto immunes ou encore d infections chroniques et chez des personnes saines En 1998 Schellekens et ses collaborateurs ont signal que les auto anticorps r actifs aux peptides synth tiques lin aires contenant de la citrulline taient tr s sp cifiques de la PR Les tudes ult rieures ont d montr que les variantes cycliques de ces peptides lin aires appel es peptides cycliques citrullin s CCP taient aussi sp cifiques de la PR mais avec une sensibilit sup rieure aux peptides lin aires Il a t montr que les anti CCP peuvent tre mis en vidence tr s t t au cours de la maladie souvent en l absence de sympt mes cliniques et un nombre important de publications indique que des taux lev s d anticorps anti CCP peuvent permettre de pronostiquer le d veloppement d une maladie rosive Ces r sultats sugg rent un r le important des anti CCP dans le diagnostic de la PR un stade pr coce de la maladie FIDIS anti CCP rreradog repose sur l utilisation de microsph res de t
33. xample 2 16 wells have already been used Use A3 as the 1st well 1 2 3 4 5 6 7 8 9 10 11 12 ASSAY PROCEDURE Microspheres dispensing Before to start the assay and if it is necessary protect the unused wells with a microplate sealing film Vortex the microspheres reagent vigorously for 20s and dispense 50uL in each well to be used PLEASE NOTE 3 If CARIS is used the microspheres should be sonicated for 5 minutes If a dispensing diluting device is used like CARIS the procedure steps 1 and 2 should be reversed gt dispense diluted sera in first and then dispense microspheres Addition and Incubation of the samples Dispense 100uL of sample dilution buffer B for reagent blank Dispense 100uL of prediluted controls of ready to use calibrator and of prediluted samples Cover the plate and incubate 30 minutes at room temperature away from direct sunlight and without shaking Wash step 1 Wash the plate 2 times with washing buffer C using filtration unit a b Remove the microplate lid verify if the vacuum break valve is closed and place it on the washer Start the pump Stop it when the whole incubation liquid has been removed in totality and open the vacuum break valve Close the vacuum break valve Dispense 300uL of washing buffer C in all the used wells Start the pump Stop it when the whole buffer has been removed and open the vacuum b
34. xtures in copper or lead piping Rinse thoroughly after flushing Avoid to use reagents if signs of contamination or other visible changes occur The FIDIS anti CCP r has been developed according to CE Directives 67 548 EEC and 1999 45 EC regarding classification packaging and labelling of dangerous preparations The FIDIS anti CCP has been optimized for use as described in this procedure Do not substitute other manufacturer reagents Dilution or alteration of these reagents may also alter the performance of the test Follow thoroughly the test procedure to insure optimal performance Calibrators and controls are from human origin The human sera used in the preparation of these products were tested and found non reactive for antibodies to HIV 1 HIV 2 anti HCV and Hepatitis B antigen Because no test method can offer complete assurance that infectious agents are absent handle as if capable of transmitting infectious diseases Vial B of sample dilution buffer white vial Ready to use neady to use DIL SPE 2x115mL Vial of calibrator Ready to use Each titer is printed on the vial label 1x1 5mL CAL Vial of positive control concentrated This control has a standard reactivity which provides evidence of the proper reagents activity and proper assay performance To be diluted 1 x 250 uL Expected values are printed on the vial label CONTROL Vial of negative control concentrate To be dilute
35. zide de sodium peut former des m langes explosifs lors de son limination dans les canalisations de cuivre ou de plomb Rincer abondamment lors de telles liminations Ne pas utiliser les r actifs si des signes de contaminations ou de modifications apparaissent Le coffret FIDIS anti CCP a t labor dans le respect des Directives europ ennes 67 548 CEE et 1999 45 CE en ce qui concerne la classification l emballage et l tiquetage des pr parations dangereuses FIDIS anti CCP a t optimis pour les conditions op ratoires pr cis es dans cette notice Le non respect des dilutions de la pr paration des r actifs du protocole ou la substitution d un r actif par un autre produit peuvent affecter les performances finales du test Les contr les et le calibrateur sont d origine humaine Pour chacun les recherches d anticorps anti HIV 1 et 2 anti HCV et d antig nes de l h patite B se sont r v l es n gatives S agissant de produits potentiellement infectieux il est toutefois n cessaire de les manipuler avec les pr cautions d usage Plaque de 96 micropuits membrane filtrante munie d un couvercle 1 plaque Flacon A de 4 cat gories de microsph res color es sensibilis es par les peptides synth tiques HCP1 HCP2 VCP1 et VCP2 Lyophilis es reconstituer avec le tampon D MICROSPHERES qsp 6ml Flacon B tampon de dilution des chantillons flacon
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