mixed withserumcontainingCRF to inactivatecomplementfactorsthatmayinterferewiththetest. The coatedparticlesagglutinatewhen specifically alteredtoreactwithcaninerheumatoidfactor(CRF).Serumsamplesareheatedinorder The latexparticlesprovidedasastandardizedsuspensioninthiskitarecoatedwithcaninelgG and jointswelling(Sodikoff, 1981). Canine rheumatoidarthritisisanerosivepolyarthritisfrequentlycharacterizedbymulti-sitelameness III. or negativereactivity. Positive andnegativereferenceseraareprovidedasacomparativeaidtotheuserinidentifyingpositive references. to eliminatetheneedforpredilutionofpatientsampleorreferences.Donotdilutesamples specialized equipmentorinstruments.Reagentandsamplevolumeshavebeenspecificallyoptimized coated onthelatexparticles. The agglutinationreactionisobservedvisually, without theneedfor II. test forthepresenceofrheumatoidfactorindogs(W Of these arethelatexagglutination,modifiedRose-Waaler, andthebentonitefloculalionprocedures. Several methodshavebeendevelopedforthedetectionofcaninerheumatoidfactor(CRF),among reported tobepresentin70%ofdogswithRA (Sodikoff, 1981). the presenceofrheumatoidfactor(RF),anantibodywhichbindstoalteredlgG,andhasbeen American Rheumatism Association (Shultz,1977)forthediagnosisofRA incanines. Among theseis The diagnosisofcanineRA isdifficult.Mostauthorshavechosentoadaptthecriteriaadoptedby osteoarthritis (degenerativejointdisease)andsepticarthritis. I. Factor For TheDetectionofCanineRheumatoid CRF ANTIGEN TEST KIT CANINE RHEUMATOID FACTOR IV .

these thelatexagglutinationprocedurehasbeendemonstratedtobeasimple,stableandconsistent 3. 2. 1. REAGENTS PROVIDED TEST PRINCIPLES INTRODUCTION 1. WARNING ® test. RFnegativereferenceserumispreservedwithGentamicinsulfateand Amphotericin B. RF negativereferenceserum,1vial.0.5mlSerumwhichisnon-reactiveinthe latexslide Amphotericin B. the latexslidetest.RFpositivereferenceserumispreservedwithGentamicin sulfateand RF positivereferenceserum,1vial,0.5ml-Serumdemonstratesreactivity in test procedure.Latexslidereagentispreservedwithsodiumazide. latex particlescoatedwithtreatedcaninelgG,standardizedtoprovideproper reactivityinthe Canine-Rheumatoid factorlatexslidetestreagent,1vial,5.0ml-A suspension ofpolystyrene The toxicologicalpropertiesofthesereagentshavenotbeendetermined.DO NOT INGEST , presentinrheumatoidarthritis,whichrecognizesandbindstoantigen The diseaseisoftenprogressiveinnatureanddistinctfrom ood, Hurvitz,andSchultz,1980). . VIII. IX. VII. Use onlycleanserumspecimens. The useofplasmaisnotrecommended.Storesamplesat2-7°C. Store componentsat2-7°C.DONOT VI. shown onthelabel. V. 3. 2. 2. 1. Materials RequiredButNotProvided 7. 6. 5. 4. 3. 5. 2. All reagentsandserumspecimensmustbeequilibratedtoroomtemperature(18°C-25°C). ASSAY 4. 3. 2. 4. 3. 1. NOTES ONPROCEDURE 2. 1. Materials Provided MATERIALS REQUIRED SPECIMEN COLLECTION STORAGE INSTRUCTIONS 1. Hand magnifier–usefulforweakspecimens Light sources– T Water Bathat56°C Stir sticks 10 μL pipets Dropper Glass ReadingSlide Negative ReferenceSerum PositiveReferenceSerum positive referencesuppliedisadjustedtoamid-rangeorapproximately2+ reactivity. indicate positivereactivity;non-agglutinatedreagentsnegativereactivity Compare serumsampleswiththepositiveandnegativereference. Agglutinated reagents under atungstenfilamentlamp(e.g., T Rotate theslideevenlyinafigureeightpatternfortwotothreeminutes.Examine with allofthesample. Stir thecontentsofeachwellwithacleanstirringstick.Makesurethatreagentmixes sample ofreferenceserum. Place 3drops(approximately120μL)ofthelatexreagentintoeachwelladjacentto reagent afewtimes. Do notallowreagenttofoam. Mix theCRFlatex-reagentbygentlyswirlingvialcontentsforabout10-15seconds. d. c. b. a. FOR INVITROVETERINARY The kitcomponentscontainsodiumazide.Upondisposalflushwithalargevolumeofwater Procedure. Do results. After washingslideindetergent,rinsethoroughly;residualdetergentmay affect test Use only Do notallowserum be separatedbycentrifugationorstandardserumfitterseparatortubes. Use onlyclearserumsamples,donotuseplasmasamples. After bloodclotting,serum may CRF PROCEDURE

® latexReagent Pipet 10μL ofserumspecimensintowellsNo.3-6. Pipet 10μL ofnegativereferenceintowellNo.2. Using thepipetsprovided,deposit10μL ofpositivereferenceintowellNo.1. Place serumsamplesin56°Cwaterbathfor30minutes. Allow samplestocoolroom temperature (70-78°F)(2125°C)beforetesting. the proportionsoflatexreagentandserumspecifiedinSlide

not predilutepatientsample. ensor lamporsimilar to drybeforeadditionoflatexreagent. FREEZE. These reagentsarestableuntiltheexpirationdate Mix thedroppercontentsbyexpellingintobulk DIAGNOSTICUSEONLY ensor orsimilar)forlatexagglutination. Test . The .

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Project No. Artwork DIR No. Component Material No. Description Country 14405 50259000 40015336 Canine Rhuematoid Factor US Dimensions Drawing No. FG Material No. DIR Type/Description 10.00” x 8.25” folds to 5.00” x 2.75” Z14-801711 10006075 Z63/Insert Additional Info: Colors: EDITOR’S COPY Black Dieline GS: DATE:

Rev GA PR GS / ART REV (LCA) GS / ART REV (FA) GS W. Floyd CHANGES CHANGES CHANGES GA S. Lewis/JD 4 OK OK OK Helpful Hints: Pipetlng 5) Symmetric joint swelling (observed by a physician) with simultaneous involvement • Using the microcapillary pipet provided in the kit: of the same joint on both sides of the body (bilateral involvement of proximal a. Insert capillary pipet into the holder provided. interphalangeal, metacarpophalangeal, or metatarsophalangeal joints is acceptable without absolute symmetry). Terminal phalangeal joint involvement will b. Allow pipet to draw up patient or reference serum by capillary action. The hole at the not satisfy this criterion. top of the rubber bulb must be unobstructed. 6) Subcutaneous nodules (observed by a physician) over bony prominences, on c. To expel the sample into a well on the glass slide, cover the hole at the top of the black extensor surfaces, or in juxta-articular regions. rubber bulb with your index linger and gently squeeze the bulb. The serum will be deposited on the slide. 7) X-ray changes typical of rheumatoid arthritis (which must include at least bony decalcification localized to or greatest around the involved joints and not just • It may be helpful to practice this technique a few times with clean water. degenerative changes). Degenerative changes do not exclude patients from any • If patient or reference serum is accidentally sucked into the glass barrel of the micropipet group classified as rheumatoid arthritis. holder, disassemble, rinse well with water, dry and reassemble before reuse. 8) Positive agglutination test - demonstration of the rheumatoid factor by any method • Do not reuse capillary pipets. which, in two laboratories, has been positive in not >5% of normal controls. • Other pipeting devices capable of delivering 10 μL and yielding satisfactory results with the 9) Poor mucin precipitate from synovial fluid (with shreds and cloudy solution). positive and negative reference sera may also be used. 10) Characteristic histologic changes in synovial membrane with three or more of the following: marked villous hypertrophy; proliferation of superficial synovial X. RESULTS cells, often with palisading; marked infiltration of chronic inflammatory cells If latex agglutination occurs in the latex slide test, rheumatoid factor is present in the test serum. Lack (lymphocytes or plasma cells predominating), with tendency to form “lymphoid of reactivity indicates the absence of rheumatoid factor. Occasionally a slight degree of agglutination nodules”; deposition of compact fibrin either on surface or interstitially; foci of cell may be encountered. Such results may indicate the presence of an early stage of progressive disease necrosis. and the test should be repeated at a later visit. Increased activity provides evidence for progressive 11) Characteristic histologic changes in nodules showing granulomatous foci disease. Generally, reactions equal to or greater than the positive reference serum indicate presence of with central zones of cell necrosis, surrounded by a palisade of proliferated rheumatoid type antibodies. Such findings combined with the criteria in section XIII assist in the diagnosis macrophages, and peripheral fibrosis and chronic inflammatory cell infiltration, of rheumatoid arthritis in the canine. predominantly perivascular. XI. LIMITATIONS OF THE PROCEDURE b. Definite Rheumatoid Arthritis This diagnosis requires five of the above criteria. In criteria 1 through 5 the joint signs or 1. Use only clear serum samples; plasma or lipemic samples may non-specifically agglutinate symptoms must be continuous for at least 6 wk. latex particles. c. Probable Rheumatoid Arthritis 2. Store the latex slide reagent with the cap tightly closed to prevent evaporation of the buffered This diagnosis requires three of the above criteria. In at least one criteria 1 through 5 liquid. the joint signs or symptoms must be continuous for at least 6 wk. 3. If the glass slide is rotated for more than five minutes, false positive results may occur. d. Possible Rheumatoid Arthritis This diagnosis requires two of the following criteria and total duration of joint symptoms XII. PERFORMANCE CHARACTERISTICS must be at least 3 wk. 1. Sera from a group of healthy dogs were found to exhibit negative reactivity when tested by 1) Morning stiffness. the Canine Rheumatoid Factor Latex Slide Test. 2) Tenderness or pain on motion (observed by a physician) with history of 2. Sera from a group of dogs exhibiting rheumatoid factor as determined by a qualified clinical recurrence or persistence for 3 wk. laboratory performing veterinary rheumatoid factor determinations were found to exhibit positive reactivity in the Canine Rheumatoid Factor Latex Slide Test. 3) History or observation of joint swelling. 4) Subcutaneous nodules (observed by a physician). XIII. HELPFUL HINTS 5) Elevated ESR or C-reactive protein. Utilizing criteria established in humans and accepted by the American Rheumatoid Association the 6) lritis (of dubious value as a criterion except in the case of juvenile rheumatoid following criteria might be helpful, as quoted from Berkow, et al.1: arthritis). 1. Diagnostic Criteria for Rheumatoid Arthritis: a. Classic Rheumatoid Arthritis XIV. REFERENCES This diagnosis requires seven of the following criteria. In criteria 1 through 5 the joint 1. Berkow, MD. R. (Editor), 1977. The Merck Manual of Diagnosis and Therapy - Thirteenth signs or symptoms must be continuous for at least 6 wk. Edition. Merck Sharp Dohme Research Laboratories-Division of Merck & Co., Inc., Rahaw, NJ. 1) Morning stiffness. 2. Schultz, A.O., 1977. Laboratory Diagnosis of Immunological Diseases. In Kirk, R. W. (Editor). 2) Pain on motion or tenderness in at least one joint (observed by a physician). Current Veterinary Therapy VI. W. B. Saunders, Co., Philadelphia, PA. 3) Swelling (soft tissue thickening or fluid, not bony overgrowth alone) in at least one 3. Sodikoff, C., 1981. Laboratory Profiles of SmallAnimal Disease. American Veterinary joint (observed by a physician). Publications, Inc., Santa Barbara, CA. 4) Swelling (observed by a physician) of at least one other joint (any interval free of 4. Wood, D. D., Hurvitz, A. I. and Shultz, R. D., 1980. A Latex test for canine rheumatoid factor. joint symptoms between the two joint involvements may not be more than 3 mo). Vet. lmmunol lmmunopathol., 1:103-111.

Zoetis Inc. Kalamazoo, MI 49007 VLN/PCN 190/5012.00 Printed in U.S.A. 40015336

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