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1. 5 minutes at 1500g Decant supernatant Resuspend pellet in 0 2 0 5mL of PBS and mix vigorously Preparation of Bronchial Wash and Bronchoalveolar Lavage Specimens Centrifuge the specimen at 1500g for 10 minutes Remove the supernatant leaving 0 5mL Decant supernatant Resuspend pellet in 0 2 0 5mL of PBS and mix vigorously PREPARATION OF SLIDES Apply 25uL to 1 well 0 8cm slide and air dry Fix slide for 5 minutes in acetone If slides are not stained immediately they may be stored below 0 C for several weeks INSTRUCTIONS FOR USE Add 25uL of monoclonal reagent to the fixed test and control slide covering well area Incubate the slides in the dark at 37 C in a humid chamber for 30 minutes Do no allow slides to dry out Wash the slides gently in PBS bath drain the slides and remove excess moisture until just dry Add 25uL of the anti mouse Ig FITC reagent RMIto the specimen Incubate slides in the dark as in step 2 Wash the slides gently in PBS bath drain the slides and remove excess moisture until just dry Add a drop of to the slide well Place a coverslip on top of the drop and remove air bubbles Scan the entire specimen using a fluorescence microscope initially at X400 magnification then at X1000 for confirmation Read immediately or store at 2 8 C in the dark for up to 24 hours READING AND INTERPRETATION OF RESULTS AND DIAGNOSIS 1 PC should exhibit bright apple green fluorescence of the round thick walled cysts and pleomorp
2. Virani N A Chaisson R E Yajko C D Sphar H T Cabrian K C Rollins N Charache P Krieger M Haldey W K Hopewell P C 1990 Rapid detection of Pneumocystis carinii using a direct fluorescent monoclonal antibody stain J Clin Micro 28 2228 2233
3. saline PBS for washing step cover slips non fluorescing immersion oil and fluorescence microscope with filter system for FITC maximum excitation wavelength 490nm mean emission wavelength 530nm and x400 x1000 magnification PRECAUTIONS For in vitro diagnostic use only Do not use after the expiry date shown on the label If protective packaging is damaged contact your local distributor and ask for a replacement Do not mix reagents from different kits The Pneumo Cel Reagent has been optimised for use with Cellabs Mounting Fluid Evans Blue dye contained in the test reagent is a possible carcinogen therefore avoid contact with the skin Patient specimens and the positive control slide should be handled as though potentially infectious A Positive Control Slide must be run with each test run Refer to Material Safety Data Sheet MSDS for further information SPECIMEN COLLECTION Clinical specimens should be processed as soon as possible after collection Induced sputum bronchial wash bronchoalveolar lavage and tissue impression smears may be used The presence of mucous in specimen may prevent adequate staining Work in a biohazard hood A positive control slide should be included with each series of test specimens Preparation of Induced Sputum Specimens Prepare 0 3 dithiothreitol DTT in distilled water Mix equal volumes of sputum and DTT solution Mix vigorously vortex to dispense clumps Incubate for 30 minutes at 37 C Centrifuge for
4. FIGURE 1 PNEUMO CEL INDIRECT DIAGRAM FOR USE Prepare specimen slide Add 25uL of to specimen slide Incubate for 30 minutes at 37 C Wash for 1 minute in PBS and remove excess moisture from slide Add 25yL of to specimen slide Incubate for 30 minutes at 37 C Wash for 1 minute in PBS and remove excess moisture from slide Add drop of and cover Scan at X4007 X1000 TABLE 1 SENSITIVITY SPECIFICITY amp OTHER DATA ON THE PNEUMO INDIRECT CEL Sensitivity Specificity Repeatability Reproducibility 0 Candida albicans EXPLANATION OF SYMBOLS pA Consult Instructions for Use In Vitro Diagnostic Medical Device Temperature Limitation 8 C 2 C LOT Batch controt Control Positive 5 Use By Expiration Date Q Do Not Re use Cellabs Pty Ltd Unit 7 27 Dale Street PO Box 421 Brookvale NSW 2100 Australia Tel 61 2 9905 0133 Fax 61 2 9905 6426 Web http www cellabs com au Email sales cellabs com au Bergen Bergerweg 18 6085 AT Horn The Netherlands LP2 3 10 December 2013 C 0843 Insert Version Not cross reactive with Cryptococcus neoformans Candida tropicalis Saccharomyces cerevesiae Aspergillus niger Candida krusei PNEUMO CEL CelLabs INDIRECT IF TEST INTENDED USE The Pneumo Cel Indirect IF Test is an invitro indirect Immunofluorescence test for the detection of Pneumocystis carinii P jirovecii in human specimens INTRODUCTION P carinii P jirovecii PC is an extracellular euka
5. ION DES LAMES D poser 25 uL sur le spot 8 mm d ne lame microscopique et laisser s cher l ir Fixer l amp c tone pendant 5 minutes Si la lame n st pas trait e imm diatement elle peut tre conserv e 0 C pendant plusieurs semaines MODE D EMPLOI 1 D posez 25 uL de RP2 sur le spot de la lame de T ou sur le spot de lame de l chantillon patient fix Incubez les lames 37 C en chambre humide pour 30 minutes Ne laissez pas les lames s cher car cela accro t le risque de marquages non sp cifiques Rincez les lames d licatement dans un bain de P B S pendant 1 minute Egouttez les lames afin d liminer tout liquide excessif jusqu ce qu lles soient s ches D posez 25 uL de sur le spot de la lame de ewa ou sur le spot de lame de chantillon patient fix Incubez les lames 37 C en chambre humide pour 30 minutes Ne laissez pas les lames s cher car cela accro t le risque de marquages non sp cifiques Rincez les lames d licatement dans un bain de P B S pendant 1 minute Egouttez les lames afin d liminer tout liquide excessif jusqu ce qu lles soient s ches REFERENCES D posez une goutte de sur chaque spot Placez la lamelle en vitant les bulles Observez le sp cimen au microscope fluorescence sous huile immersion initialement x400 puis x1000 pour confirmation Si l bservation est retard e conservez les lames I bscurit 2 8 C jusqu 24 heures Images of human Pneumocystis carinii
6. a ae n treated with cyclosporine and steroids J Infect Dis 149 143 147 observable Ignorez tout l ment fluorescent qui n st pas vert pomme et qui se distingue de la forme typique de P carinii 5 Hughes W T 1987 Pneumocystis carinii pneumonia N Eng J Med 317 1021 3 DECHETS 6 Kovacs J A Masur H 1988 Pneumocystis carinii pneumonia therapy and prophylaxis J Infect Dis 158 254 9 7 Murray J F Garay S M Hopewell P C Mills J Snider G L Stover D E 1987 Pulmonary complications of the acquired immunodeficiency syndrome an update report of the Second National Heart Lung and Blood Institute Workshop Am Rev Respir Dis 135 504 509 8 Phair J Munoz A Detels R Kalslow R Rinaldo C Saarh A 1990 The risk of Pneumocystis carinii pneumonia among men infected with the human immunodeficiency virus type 1 New Engl J Med 322 161 165 Jetez tout composant inutilis dans la poubelle aux d chets biologiques Lorsque vous videz le r actif du coffret dans I vier assurez vous dans le diluer avec une large quantit d amp au courante car l amp zide de sodium qud contient peut tre explosif en contact avec les gouts en cuivre ou en plomb Consultez la fiche de s curit du produit notice MSDS pour plus amples informations SENSIBILIT SP CIFICIT ET AUTRES DONNEES DU TEST PNEUMO CEL 9 Pilon V Echols R Celo J Elmendorf S 1987 Disseminated Pneumocystis carinii pneumonia infec
7. ese techniques are rapid easy to perform and specimens from induced sputum and bronchoalveolar lavage can be used thus avoiding the need for more invasive procedures such as lung and transbronchial biopsies IFA has been shown to be both specific and sensitive 12 13 and also more sensitive than histochemical staining 14 The PNEUMO CEL INDIRECT IF TEST is an indirect immuno fluorescence test for PC organisms in methanol fixed specimens The fluorescein labelled mouse monoclonal antibodies bind specifically to the murine raised monoclonal initially bound to the organisms Washing steps following both the monoclonal and fluorescein conjugated antibodies removes any unbound antibody When viewed under a fluorescence microscope PC are seen as bright apple green bodies which may be present singularly or in cluster Trophozoites cysts and the extra cellular matrix which binds cysts are stained CONTENTS OF THE KIT KP2 Standard Bulk RP2 Monoclonal Antibody Reagent 1 25mL 5mL Anti Mouse FITC Reagent 1 25mL 5mL RMF Mounting Fluid 2 5mL 2 x 2 5mL Tests 50 200 Materials are supplied ready for use Store at 2 8 C Expiry dates are clearly marked on each kit component and on the box Expiry dates do not change once opened MATERIALS REQUIRED BUT NOT PROVIDED Positive control human PC microscope slides with 6 8mm diameter wells precision pipette for delivering 25uL methanol for specimen fixation humid chamber wash bath phosphate buffered
8. hic trophozoites in contrast to the reddish brown colour of counterstained material The organisms occur either singularly or more commonly in clusters in an extracellular matrix which is also stained A test is considered positive if two or more cysts are present 3 Negative specimens should not display fluorescent organisms of characteristic morphology 4 Fluorescent material which is not apple green and can be distinguished from typical PC forms should be disregarded WASTE DISPOSAL Dispose of any unused components as biohazardous waste Where the test reagent has been disposed of in the sink ensure it is flushed with large quantities of water as the sodium azide it contains may react with copper lead plumbing systems For more information please refer to the MSDS SENSITIVITY SPECIFICITY amp OTHER DATA ON THE PNEUMO CEL Refer to summary table at end of insert All data on the Pneumo Cel can be obtained in the product information sheet Please ask your local distributor or contact Cellabs INDEMNITY NOTICE Modifications or changes made in the recommended procedure may affect the stated or implied claims A positive or negative result does not preclude the presence of other underlying causative agents Cellabs and its agents and distributors shall not be liable for damages under these circumstances NOOR WN gt PNEUMO CEL INDIRECT PRINCIPE DU TEST ET INDICATIONS D EMPLOI Le coffret Pneumo Cel IF est un test in vitro par immunoflu
9. orescence directe con u pour d tecter et diagnostiquer la pr sence de Pneumocystis carinii dans les chantillons humains L nticorps monoclonal de souris marqu la fluoresc ine se lie sp cifiquement aux P carinii de chantillon P carinii fluorescent dain vert intense et d montrent leur morphologie typique COMPOSITION DU COFFRET KP1 Standard Bulk RP2 R actif Pneumo Cel 1 25mL 5mL Anti Mouse FITC Reagent 1 25 mL 5mL RMF Liquide de Montage 2 5mL 2 X 2 5mL D terminations 50 200 Conserver 2 8 C Les dates de p remption sont clairement indiqu es sur chaque composant et sur I tiquette du coffret et ne sont pas affect es par uverture du coffret MATERIELS REQUIS NON FOURNIS Lames a spots diam tre 6 8 mm pipette de 25 uL ac tone pour fixation des chantillons chambre humide bain de lavage des lames tampon P B S pour lavage lamelles couvre objets huile a immersion non fluorescente microscope a fluorescence pour FITC 490 530 nm grossissement 400 a 1000 PRECAUTIONS Produit usage uniquement in vitro Ne pas utiliser apr s la date de p remption indiqu e sur l tiquette Si I amp mballage est abim contactez votre fournisseur local pour un remplacement Ne pas m langer les composants de coffrets diff rents Pneumo Cel est optimis pour l amp mploi des lames de contr le et du liquide de montage fournis par Cellabs Le Bleu d amp vans contenu dans le r actif est un carcinog ne potentiel donc vitez tout con
10. ryotic organism shown to be related to the fungi 1 It is an opportunistic pathogen causing interstitial pneumonia in immunosuppressed hosts such as premature infants children with congenital immunodeficiencies and organ transplant recipients 2 3 4 With the advent of the acquired immunodeficiency syndrome AIDS P carini P jirovecii i pneumonia PCP has emerged as the most common life threatening opportunistic infection in these patients PCP occurs in over 75 of AIDS patients 5 6 7 and is usually associated with diminished CD4 cells 8 PC is generally confined to the lungs although there have been recent reports of disseminated extrapulmonary disease 9 which may be as a result of the common use of aerosolised pentamidine 10 The most common form of PC is the thick walled cyst 5 8um containing up to 8 intracystic bodies sporozoites 1 2um These mature into extracystic pleomorphic forms the trophozoites 2 5um The development cycle occurs in both the alveolar epithelial cells and in the alveolar spaces PRINCIPLE OF THE TEST PC may be identified by a variety of histochemical stains such as methenamine silver Giemsa and toluidine blue 11 The stains can however be difficult to perform they are non specific and they may not detect all stages of the life cycle of the organism Experience is also required for interpretation More recently immunofluorescence assays IFA using monoclonal antibodies to PC have been developed Th
11. stained with Pneumo Cel Indirect IFA ONDARON 1 Edman J C Kovacs J A Masur H Santi D V Elwood H J Sogin M L 1988 Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi Nature 334 519 22 OBSERVATION INTERPRETATION DES RESULTATS ET DIAGNOSTIQUE 2 Walzer P D Schultz M G Western K A Robbins J B 1976 Pneumocystis carinii pneumonia and primary immune deficiency diseases A AR A re z Nat Cancer Inst Monogr 43 65 72 A Inverse de la coloration de fond brun rouge tre les trophozoites pleomorphes de Pneumocystis carinii PC pr sentent une intense fluorescence vert pomme alors i Le que les cystes a paroi paisse pr sentent une fluorescence en halo autour de leur p riph rie Les organismes peuvent tre seuls ou plus fr quemment en grappes 3 aa a Penk Pare oe EE L J eles S maiak aboni N To an Nemes te ieee ri 1988 incluses dans une membrane extracellulaire aussi color e Lorsque vous observez au moins deux cystes le r sultat est positif Utilisez la lame de contr le titre de i y AN H 19 spa rondin AF e A Ru rie oan poise anta ae Les ak at nie PEE T comparaison pour v rifier I pparence et la taille des organismes Le r sultat est n gatif quand aucun organisme fluorescent de morphologie caract ristique n st Hardy A M Wajszczuk C P Suffrendini A F Hakala T R Ho M 1984 Pneumocystis carinii Pneumonia in renal transplant recipients aes
12. tact avec la peau Les lames de contr le positives ainsi que les chantillons patients doivent tre consid r s comme potentiellement infectieux Une Lame de Contr le Positive doit tre pr par e pour chaque s rie de tests afin d n v rifier la qualit Consultez la fiche de s curit du produit notice MSDS pour plus amples informations COLLECTION DES ECHANTILLONS Les sp cimens cliniques doivent tre analys s d s que possible apr s leur pr l vement On peut utiliser du sputum induit du lavage bronchial ou broncho alv olaire ou des impressions de tissu sur lame La pr sence de mucus dans l amp chantillon peut emp cher une coloration ad quate Travaillez sous hotte flux laminaire Une lame de contr le doit tre incluse avec chaque s rie de sp cimens analyser Pr paration de sp cimens de sputum induit Pr parer une solution 0 3 de DTT dithiothreitol l amp au distill e M langer sputum et solution DTT volume gal M langer vigoureusement vortex pour mulsionner la solution Incuber 30 minutes 37 C Centrifuger 5 minutes 1500g D canter le surnageant Suspendre le culot dans 0 2 0 5 mL de P B S et m langer vigoureusement Pr paration de sp cimens de lavage bronchial ou broncho alv olaire Centrifuger le sp cimen 10 minutes 1500g Jeter le surnageant en en laissant 0 5 mL D canter le surnageant restant Suspendre le culot dans 0 2 0 5 ml de P B S et m langer vigoureusement PREPARAT
13. tion in AIDS N Eng J Med 316 1410 Referez vous au tableau r capitulatif en fin de notice Toutes les donn es sur le test Pneumo Cel sont sur la fiche technique du produit Contactez Cellabs ou votre 1411 distributeur pour l btenir 10 Raviglione M C 1990 Extrapulmonary pneumocystosis the first 50 cases Rev Infect Dis 12 1127 1138 NOTICE D INDEMNITE 11 Ruskin J 1986 Newer developments in diagnosis and treatment of Pneumocystis infections Curr Clin Top Infect Dis 7 194 215 Toute modification ou variation du protocole d amp mploi recommand peut affecter les performances annonc es du produit Un r sultat positif ou n gatif n xclue pas la 12 Kovacs J A Ng V L Masur H Leoung G Hadley W K Evans G Lane H C Ognibene F P Shelhamer J Parillo J E Gill V J 1988 pr sence d utres agents causatifs sous jacents Cellabs et ses agents et distributeurs ne sont l galement responsables d ucun dommage dans de telles Diagnosis of P Carinii pneumonia improved detection in sputum with use of monoclonal antibodies New Eng J Med 318 589 593 circonstances 13 Wolfson J S Waldron M A Sierra L S 1990 Blinded comparison of a direct immunofluorescent monoclonal antibody staining method and a Giemsa staining method for identification of P Carinii in induced sputum and bronchoalveolar lavage specimens of patients infected with human immunodeficiency virus J Clin Micro 28 2136 2138 14 Ng V L
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