Protocol For Postmortem Diagnosis Of Rabies In Animals By .
Protocol for Postmortem Diagnosis of Rabies in Animals by DirectFluorescent Antibody TestingA Minimum Standard for Rabies Diagnosis in the United StatesI. IntroductionAmong the findings of the National Working Groupon Rabies Prevention and Control was the needfor a minimum national standard for thelaboratory diagnosis of rabies (Hanlon et al.,JAVMA, 215:1444-1446, 1999). In response tothis recommendation, a committee was formedfrom representatives of national and state publichealth laboratories (Appendix 1) to evaluateprocedures employed by rabies diagnosticlaboratories in the United States. Both theNational Working Group and this committee haveas their goal the improvement of the overallquality of rabies testing through the formulationof guidelines and standards for equipment,reagents, training, laboratory protocols, qualityassurance, and laboratory policy for rabiesdiagnosis.As a first step to attaining this outcome, thecommittee prepared a standardized protocol forthe analytical phase of rabies testing using thedirect fluorescent antibody (DFA) test andevaluated the protocol by comparison testing of435 samples submitted to public healthlaboratories for rabies diagnosis (Appendix 2) .Later documents will address other elements thatcan affect the quality of laboratory testing such asthe pre-analytical steps (rationale for specimensubmission), the post-analytical phase (resultreporting and confirmatory testing), laboratorypolicy, and quality assurance.The standardized protocol was developed frompublished procedures and the collective laboratoryexperience of the committee members. The grouprecognizes that a range of possible methods mayachieve the desired outcome for some of the lesscritical steps in the diagnosis of rabies and thatlaboratory policy may be regionally defined insome cases; however, the goal of the group wasto establish a single protocol by which all othermethods could be validated by comparison. Therecommendations included in this documentshould be closely followed to ensure a test ofhighest sensitivity and specificity. Modifications orshort cuts in procedures often lead to falsepositive and false negative results and non specific or uninterpretable reactions. A laboratorywishing to incorporate elements of test methodsother than those presented in this documentshould validate and confirm those methods byconsultation with one of the laboratories listed inAppendix 1.The standard protocol for DFA will be madeavailable to each rabies testing laboratory bypostal or electronic mail and by participation in atraining workshop. In addition, the protocol andother documents will be placed on the web sitesmaintained by the Centers for Disease Control andPrevention (CDC) at www.cdc.gov/rabies and theAssociation of Public Health Laboratories (APHL)at www.aphl.org. A listserve is planned so that aninteractive dialogue may be established toaddress questions and disseminate informationabout rabies testing.In addition to the procedural aspects detailed inthis document, each testing laboratory mustmaintain the competency of its employees. Thereis no substitute forconstant practice and experience in performingDFA testing. All new employees should be trainedin all aspects of the procedure, and competencyshould be evaluated by the senior technologist ona routine basis. Competency can be assessed byobservation of all procedural aspects on a routinebasis, as well as performance on proficiency testsamples, and testing on internal blind samples. Alltraining should be documented throughout thetraining period, and observations of breakdown intechnique or procedures should be noted andcorrected before the lead technologist can beassured that the trainee can perform theprocedure in a competent and reliable manner. Alllaboratories performing this DFA test for rabiesshould participate in national rabies virusproficiency testing, available through theWisconsin State Laboratory of Hygiene.Enrollment information may be obtained throughtheir web site (www.slh.wisc.edu/pt) or by calling1-800–462–5261.At least once every 6 years, each laboratoryshould send a representative to the NationalLaboratory Training Network sponsored course"Laboratory Methods for Detecting Rabies Virus".The course, held at least every 2 years, details allaspects of rabies testing and provides anopportunity for diagnosticians to meet colleaguesfrom other states and discuss common problemsand their solutions. Advances in rabies diagnosisand research are presented at the Rabies in theAmericas meeting, held every year at locations inNorth and South America.Bench training and consultation on basic aspectsof rabies diagnosis are available at the state
public health laboratories in New York, California,Texas, Wisconsin, and Ohio and at the nationalrabies laboratory at CDC (Appendix 1). Theselaboratories can be contacted at any time withquestions or requests for consultation andtraining. Laboratories that annually process 100samples may have particular difficulty maintainingrabies diagnostic proficiency and may want towork closely with larger laboratories whereadditional resources are available. Sources offunding are being investigated for those statesthat have no travel budget to attend meetings,workshops, or seminars. Potential sources thatare being explored include ASM, APHL, or theEpidemiology and Laboratory Capacity (ELC)program at CDC.II. Safety All persons involved in rabies testingshould receive pre-exposure immunization withregular serologic tests and booster immunizationsas necessary (CDC, MMWR, 48: 1-22, 1999).Unimmunized individuals should not enterlaboratories where rabies work is conducted. Alltissues processed in an infectious diseaselaboratory must be disposed of as medical wasteand all activities related to the handling of animalsand samples for rabies diagnosis should beperformed using appropriate biosafety practices toavoid direct contact with potentially infectedtissues or fluids (CDC and National Institutes ofHealth, Biosafety in Microbiological andBiomedical Laboratories, 4th edition, U.S.Government Printing Office, 1999). Personnelworking in rabies laboratories are at risk of rabiesinfection through accidental injection orcontamination of mucous membranes with rabiesvirus contaminated material and by exposure toaerosols of rabies infected material. Allmanipulations of tissues and slides should beconducted in a manner that does not aerosolizeliquids or produce airborne particles. Barrierprotection is required for safe removal of braintissue from animals submitted for rabies testing.At a minimum, barrier protection during necropsyshould include the following as Personal ProtectiveEquipment (PPE): heavy rubber gloves, laboratorygown and waterproof apron, boots, surgicalmasks, protective sleeves, and a face shield.Fume hoods or biosafety hoods are not required,but they provide additional protection from odor,ectoparasites, and bone fragments. Glass chipsand shards from slide manipulations are alsopotential sources of exposure to rabies. Careshould be taken to protect eyes and hands duringmanipulation and staining of slides and duringclean up of the microscope and surrounding area.A microscope adaptor is available to provide eyeprotection from any glass slivers produced whenslides are moved across the microscope stage.Ergonomic equipment (fatigue mat, microscopecontrols) should be used to prevent fatiguerelated injuries to employees during lengthynecropsy and slide-reading procedures.III. Equipment and Reagents(Use of trade names and commercial sources isfor identification only and does not implyendorsement.)A. Equipment1. Necropsy instruments should be of sufficientquantity for 1 set per sample to prevent crosstransfer of infected tissue between samples.2. Autoclave and/or instrument sterilizer. Allinstruments should be cleaned and sterilizedbefore reuse.3. Specimen storage containers must be largeenough that reserved portions of brain stem andcerebellum (and hippocampus, if tested) remainas recognizably separate pieces. Because of therisk of breakage, glass vials and tubes areunacceptable for specimen storage. Wide mouth,screw cap, polypropylene jars or sample bottlesare used in many laboratories (e.g., Nalgene 2118or 2189).4. Refrigerated storage. An explosion proof 20 C freezer is required for fixation ofimpression / smear slides and storage of acetoneand other reagents; long term sample storagerequires a freezer at -70 C. Frost-free freezersshould not be used. Heat cycles in frost-freefreezers will denature proteins in reagents andspecimens and may compromise test results.5. Microscope slides should be of highestquality with coverslip matched to the lens workingdistance. Teflon or pre-ringed well slides can beused to denote stained areas for touch impressionslides. User-specified templated slides can beordered through Cel-Line/ERIE Scientific Co. (1 800-258-0834). Wells of 14 mm or 15 mmdiameter are adequate for rabies tests. Markinginstruments that contact tissue (e.g., wax pencilor Martex pen) should not be used to denotestained regions of the slides, because this processcan transfer infected tissue between slides. Slipsmears cannot be made on pre-ringed slides. A 16mm area to be stained on a slip smear can bemarked by dipping the rim of a 16 x 100 mm testtube into a small pool of nail polish poured onto asquare of aluminum foil. A well is made on theslip smear by lightly touching the test tube rim tothe surface of the smear. To avoid crosscontamination between specimens, a separatetest tube and pool of nail polish is used for eachspecimen submitted for testing. Best contrast isobtained with red pigmented nail polish.Alternatively, separate slides can be made for
staining with each reagent. The addition of acounter stain such as Evans Blue to the conjugatewill also denote stained areas.6. Acetone fixation and post-stain rinsecontainers should be in sufficient quantity suchthat slides made from each test animal areprocessed in a container separate from the slidesfrom other animals. Positive control slides areprocessed in a container separate from test clides.A false-positive test may result from crosstransfer of tissue between slides from differentanimals if a common container is used for fixationor washing.7. Syringe filters (0.45um). Anti-rabiesconjugates should be filtered to removedissociated fluorescein isothiocyanate (FITC) andprotein aggregates that may bind non-specificallyto tissue. Conjugates should be filtered only onceand should be evaluated for the effect of filtrationon the titer of the working dilution (see sectionVI). Syringe filters allow the conjugates to befiltered as they are added to test slides. Similarly,syringe filters will remove contaminants andprecipitates as mountants are added to slides.Filters for the conjugate must be low proteinbinding (e.g., cellulose acetate) to prevent loss oflabeled antibody from the conjugate. Filtermaterials that bind proteins with great avidity(e.g., mixed cellulose esters) should not be usedfor conjugate filtration. Both large volume, midvolume, and small volume filters are available(Schleicher & Schuell, Uniflo; Millipore, Millex-HV).Smaller volume filters avoid dead volume loss tothe filter membrane. Evans Blue counterstain alsobinds to the membrane, but the filter is saturatedquickly. To avoid slide to slide difference incounter stain, laboratories that employ Evans Bluecounterstain in the conjugate should discard thefirst three drops of conjugate expressed thoughthe filter.8. Incubator (37 C) and humidified stainingtray or chamber. Constant humidity must bemaintained during the staining process. Conjugatedried on the slides during the staining processmay be mistaken for specific staining, resulting ina false positive test, or may obscure specificstaining, resulting in a false negative test.9. Fluorescence microscope. The quality of thefluorescence microscope is critical to thesensitivity of the DFA test. Manufacturers offermany equipment options. Appendix 3 contains adiscussion of oculars, objective lens, filter sets,and light sources and their effects on instrumentperformance. At a minimum, all rabies diagnosticlaboratories should have a reflected light (incidentlight) fluorescence microscope with high qualityobjective lenses. Both magnification and andnumerical aperture (NA) must be considered inlens selection. Although image size increases withmagnification, both resolution and imagebrightness are related to the NA of the objectivelens, and brightness decreases with magnification.For example, a high quality 20X dry objective witha NA of 0.75 provides a brighter image over alarger field of view with no loss of resolution ascompared to a dry 40X objective with an NA of0.75. (See the discussion of magnification, imagebrightness and resolution in Appendix 3.) The useof immersion oil increases image brightness bypreventing the loss of emitted light in the airspacebetween coverslip and dry objective. Although notevery slide must be observed with a 40X oilobjective of high NA, resolution of very fine dustlike inclusions and recognition of some types ofnon-specific staining is aided by examination withthis type of lens. An oil immersion lens requires ahigh quality immersion oil. An oil should bechosen that produces the least autofluorescenceand thus the best contrast between FITC andtissue (e.g.,Cargille type DF). The oil should havethe same refractive index as glass (1.515).B. Reagents1. Acetone. Only American Chemical Society(ACS) Reagent Grade acetone should beemployed and the acetone should not be reused.2. FITC-conjugated anti-rabies antibodies.Reagents presently available commercially arelisted in Appendix 4. Every laboratory shouldmaintain stocks of anti-rabies conjugates fromtwo different sources. The two sources should betwo different monoclonal antibody pools or onemonoclonal antibody conjugate and onehyperimmune serum conjugate. (Note: CentocorFITC-Anti-rabies Monoclonal Globulin and LightDiagnostics Rabies DFA II contain the sameantibody pools and are not considered differentsources.) Antigen presentation and antibodyavidity and affinity vary with different rabies virussamples, and viral inclusions may appear quitedifferent when stained with different reagents.Although hyperimmune serum reagents containmany different anti-rabies antibodies that can thatproduce a wide-spectrum of rabies virusrecognition, these reagents also containextraneous antibodies that can produce severaltypes of non-specific reactions (Appendix 5).Extraneous antibodies are not a problem withmonoclonal antibody reagents but the finespecificity of these reagents may result in non recognition of some variants of rabies virus. Theuse of two reagents prepared from different poolsof monoclonal antibodies greatly reduces the riskof non-recognition of any one variant.FITC-labeled conjugates should not be used past
the manufacturer's expiration date. Upon receipt,a conjugate should be reconstituted (iflyophilized) and titrated to determine a workingdilution (section VI). Stock solutions of conjugateare stored as frozen aliquots in a non-frost-freefreezer at -20 C or below ( -30 C is preferred).Conjugate diluted to the working dilution is storedat 4 C and discarded after 7 days. The workingdilution of the conjugate is filtered as it is addedto the test slide. Excess diluted conjugate can bestored at 4 C in the syringe and attachedsyringe-filter unit used for dispensing it, if care istaken to remove any remaining droplets from thefilter outlet and the outlet is sealed against dryingwith a syringe tip or plastic wrap.3. Specificity controls. Appendix 4 listscommercially available control reagents. Thespecificity control for an FITC-labeledhyperimmune serum rabies reagent is an FITClabeled serum reagent produced in the sameanimal host as the rabies reagent (typically goator horse), but either as normal serum orhyperimmune serum directed to an agent otherthan rabies virus (e.g., FITC-labeled antidistemper reagents). The control reagent shouldbe diluted to the same mg protein concentrationas the rabies reagent. Similarly, the specificitycontrol for a rabies reagent prepared from amouse monoclonal antibody is an FITC-labeledmouse monoclonal antibody of the same isotypeand protein concentration as the rabies reagentbut directed to an agent other than rabies virus.Suspensions of Normal Mouse Brain (NMB) andRabid Mouse Brain (RMB) are no longer used asspecificity controls. Historically, RMB and NMBwere used as diluents for FITC-labeledhyperimmune serum conjugates to control for thepresence in the conjugate of extraneousantibodies present in animals used for theproduction of rabies hyperimmune serum as aresult of a natural exposure. False positivereactions were identified when staining wasobserved both with a conjugate adsorbed withNMB (which should have no effect on anyantibody in the conjugate) and a conjugateadsorbed with RMB (which should remove onlythe rabies antibodies and have no effect onantibodies to other infectious agents). Becausemonoclonal antibody reagents contain onlyantibodies reactive with antigens on rabies virus,it is not possible for these reagents to containextraneous antibodies and adsorption controls forthese reagents are meaningless. A more lengthyexplanation of control reagent use is found inSection VII and in Appendix 5.4. Conjugate diluent should be 0.01Mphosphate buffered isotonic saline solution at pH7.4 to 7.6 (e.g., phosphate buffered saline (PBS)Sigma P3813 is 0.01 M phosphate buffer, pH 7.4,with 0.138 M NaCl and 0.0027 M KCl). No proteinstabilizer (e.g., bovine serum albumin) is neededin the diluent.5. Counterstains added to the working dilutionof the conjugate provide contrast and lowerbackground and also serve as a marker foraccidental omission of the diagnostic reagent.Counterstain use is optional. Evans Bluecounterstain (0.5% in PBS, Sigma, Product #E 0133, for use in immunofluorescent assays) canbe aliquoted and stored at 4 C for up to 6months and indefinitely at -20 C. The amount ofcounterstain added to a conjugate is determinedby titration when the working dilution of theconjugate is determined (section VI). Due tocounterstain, the tissue will be noticeably red, butshould not be so strongly red as to diminish thespecific green fluorescence of rabies virusproteins. An Evans Blue concentration of0.00125% works in many laboratories. Thisconcentration is prepared by adding 2.5microliters of 0.5% stock dye solution per ml ofconjugate diluent.6. Rinse / soak buffer. A PBS formula of thesame pH and molarity as the conjugate diluent isused as the rinse/soak buffer (e.g., Sigma P3813is 0.01 M phosphate buffer, pH 7.4, with 0.138 MNaCl and 0.0027 M KCl). Carboys or other largecontainers used for storage and dispensing ofrinse buffers should be disinfected by autoclavingon a regular schedule.7. Mountant ( 0.05 M Tris-buffered saline pH9.0 with 20% glycerol). Prepare 0.05 M Tris /0.15 M NaCl solution by dissolving 0.623 grams ofTrizma pre-set crystals and 0.85 g NaCl in a totalvolume of 100 ml distilled water. Filter (0.45 um)and store at room temperature. Remake at leastonce per year; check pH quarterly. Prepare a onemonth supply of mountant by mixing 4 parts Tris saline pH 9.0 with 1 part glycerol. Store at roomtemperature. Glycerol concentrations above 20%affect the antigen binding capacity of someantibodies and should not be used. Glycerolshould be replaced at yearly intervals because thepH changes slowly with time. The mountantshould be remade or the pH tested once a month.The mountant is added to the coverslip bydispensing with a syringe fitted with a 0.45 umsyringe filter. (Trizma pre-set crystals, Sigmacatalog # T6003 ; ACS Reagent Grade glycerol,Sigma catalog #G7893)8. Immersion oil should be formulatedspecifically for fluorescence applications (e.g.,Cargille DF). Immersion oils formulated forgeneral microscopy may produce significant autofluorescence.
IV. Sample Co
Protocol for Postmortem Diagnosis of Rabies in Animals by Direct Fluorescent Antibody Testing A Minimum Standard for Rabies Diagnosis in the United States I. Introduction Among the findings of the National Working Group on Rabies Prevention and Control was the need for a minimum national standard for the laboratory diagnosis of rabies
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