4 blood sampling of finfish

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  • CanadaDepartmentofFisheriesandOceansAnimalUserTrainingTemplate

    BloodSamplingTemplatepreparedSeptember2004. Page1 of 15

    4.0BloodSamplingofFinfish

    4.1Introduction:ThistemplateisintendedforusebyinstructorstotraintheDepartmentofFisheries&Oceans(DFO)staffandstudentsinthebloodsamplingoffinfish.Templatesareusedtoprovidetheminimumrequirementsnecessaryinatrainingexercise,buttheinstructormayaddadditionalmaterialasdeemednecessary.

    Anexperiencedinstructormustdemonstratethemethodsoutlinedinthistemplateandtraineesmustbedeemedqualifiedincarryingoutoneormore oftheproceduresbeforetheyarepermittedtobloodsamplewithoutaninstructorpresent.HandsontrainingofstaffisarequirementforfacilityapprovalbytheCanadianCouncilonAnimalCare(CCAC),ofwhichDFOisamember. Thistemplateispartof acomprehensiveDFOScienceBranchseriesontrainingforusersofaquaticresearchanimals.

    4.2Rationale:Scientificstudyoftenrequiresthatfishbebloodsampledforreasonssuchashaematology,clinicalchemistryparameters,bacteriology,parasitologicalinvestigation,packedcellvolume,etc.Investigatorshaveanethicalobligationtominimizethepainand/ordistressofalllaboratoryanimalsundergoinglaboratorysamplingtechniques.OnlythosemethodsdeemedacceptablebytheCCACshouldbeemployed.

    4.3Authority:Thestaff/consultantveterinarianorAnimalCareCommitteeisresponsibleforprovidinginformationaboutbloodsamplingmethodsofthefishspeciesusedforscientificstudyintheirrespectiveregions.TheAnimalCareCommitteemaydelegatetrainingoftheseprocedurestoaninstructorwhohasdemonstratedknowledgeandexperienceintheareasofanatomyandanaesthesiaandhasdemonstratedexcellentsurvivaloffishthathavebeenbloodsampledusingthetechniquesoutlinedinthistrainingtemplate.Staffmustbetrainedintheproperbloodsamplingtechniquesforthefishspeciesandsizebeingstudiedpriortoinitiationofanybloodsamplingprocedure(s).

    4.4Goalofthistrainingexercise:1. Learnthemethodstohumanelyremovebloodsamplesfromanaesthetized

    fish.2. Understandtheconsequencesofeachmethodofbloodsamplingonthefish

    andthusthepotentialimpactofthemethodselectedandthesamplecollected.3. Understandthefunction/physiologyofthecirculatorysystem withinafish.4. Demonstrategentlehandlingtechniquesoffishwhenlivefishareusedinthis

    section.5. Understandproperdisinfectiontechniquestouseaftercompletingthe

    procedure(providebiosecuritySOPs).

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    4.5TheoreticalTrainingtobecompletedbeforehandsonsession1. CompletedTheExperimentalFish.2. CCACguidelines:SectionHExperimentalProcedures6:Collectionofbody

    fluids.3. Completedtheanatomy&physiology,anaesthesiaandeuthanasiatemplates

    orhasexperienceortraininginallthreeareas.4. WHMIStraining.5. Summarytheorymaterialprovidedwiththistrainingtemplate(AppendixA:

    Fishbloodandthecirculatorysystem).

    4.6DetailsoftheProcedure:Introductory:Methodsofbloodsampling,thatmaybeincludedinthetrainingsession,are:1. Bloodsamplingbytailablationofsmallfishthathavebeeneuthanized.2. Bloodsamplingbycaudalvenouspuncture.3. Bloodsamplingbydorsalaortapuncture.

    Advanced:4. Bloodsamplingbyheartpuncture.

    4.6.1. TimeestimateforcompletingthehandsonexerciseSetup:1hourInstructionandTraining:2hours

    4.6.2 EquipmentRequired: Fish:numberwilldependonthenumberoftraineesinasession

    speciesusedwilldependonavailabilityandrequirementsofthefacility.

    TMS(plusNaHCO3bufferifanaesthetizinginfreshwater) Dipnet Containerforanaestheticbath Airstonesandcompressedairoroxygen Thermometer Glovesandsplashglassestobewornwhenhandlingfishor

    anaesthesiawater Disinfectantforcleanupafterbloodsampling Softspongeormattopreventmucoussloughingduringblood

    sampling Scalpelorsharpknifefortailablation Papertowelforcontainmentofbloodfromablation Sterileneedlesorvacutainerneedles(sizewillbedependentonthe

    sizeofthefish) Syringeorvacutainertubeforcollectingbloodsamples(sizewill

    dependontheamountofbloodtocollectandthesizeofthefish) Vacutainerholder

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    BloodSamplingTemplatepreparedSeptember2004. Page3 of 15

    Tuberacksforbloodcollectiontubes Hematocrittubes Critoseal Sharpscontainer

    4.6.3Procedure:Theinstructorshoulddemonstratealltheprocedurespriortoallowingthetraineestoattemptthem.Fishsacrificedintheeuthanasiatemplatemakeexcellentteachingspecimensforthetraineestopracticetheirtechniques.

    Instructorswillneedtousetheirownbestjudgementinchoosingwhichproceduresareappropriatewiththecommonspeciesusedattheirresearchfacility.Itisrecommendedthatallproceduresbetaughttotraineessothattheyhavemultipleoptionsattheirdisposalwhenfacedwiththeneedtosamplethebloodoffishofdifferentspecies.

    Introductory:4.3.6.1EquipmentforBloodCollection:

    HumanSafetyMeasures Discusshumansafetymeasureswhilediscussingtheequipmentused

    forbloodsampling.

    Wearglovesatalltimeswhenhandlingbloodcollectionequipment.Whilethiswillnotpreventaccidents,itmayreducetheriskofcontamination.

    Neverattempttoresheathneedles.Alwaysdiscardthemintoasharpscontainerimmediatelyafteruse.

    Neveroverfillthesharpscontainer.Adheretotherecommendedfilllineonthesharpscontainer.

    Placethesharpscontainerintheareayouareworkingtoavoidtransportingusedneedlesoutsideofyourworkarea.

    Ensuretheareayouareworkingiscleanandalltheequipmentneedtocarryouttheprocedureisreadilyavailable.Allowenoughworkspacetosafelyobtainthebloodsamplewithoutcontaminatingthebloodsamplingsuppliesshouldthefishexhibitcommonreflexmovementsuponneedleinsertion.Forbloodcollectioninthefielditissuggestedtospreadafinemeshnetundertheentireworkareatopreventlossofsupplies(particularlysharps)thatmayaccidentallydropwhilesampling.

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    EquipmentOptions:

    Presentequipmentoptionsavailableforbloodcollection(i.e.needlesofvariousgaugesandlength,multiplesyringetypesandsizes,haematocrittubesandcritoseal,vacutainerneedlesandtubes,etc.).

    Emphasizetherelationshipbetweenneedlegaugeandneedlesize(e.g.a20gaugeneedleislargerthana30gaugeneedle).

    Discusssafetyequipmentandhowitistobeused(i.e.tuberacksareforholdingtubesafterfillingwithblood,sharpscontainersarefordiscardingusedneedles,etc.).

    Identifytheproperneedlegaugeandlengthrequiredforbloodsamplingthefishusedinthistrainingsession.

    Discussthecommontypesandsizesofvacutainertubesandchosethetypeandsizeappropriateforthefishusedinthistrainingsession.Traineesshouldconsultwiththeprincipalinvestigatortodeterminetheappropriatetubestousewhenbloodsamplesarecollectedforresearchpurposes.Examplesofvacutainertubesinclude,butarenotlimitedtothefollowing:

    Redtop:containsnoadditives.Usedforserumcollection,bacteriology,andparasitologicalexamination.

    Greentop:containstheanticoagulantlithiumorsodiumheparin.Intendedforclinicalchemistryparametersandplasmacollection.

    Purpletop:containstheanticoagulantEDTA.Intendedforwholebloodcollectionforhaematology.EDTAwillinterferewithsometestsandisthereforenotasuitablesampletubeforbloodchemicalanalysis.

    Greytop:containstheadditivesodiumfluoride.Intendedforglucosemeasurements.

    Lightbluetop:containstheadditivesodiumcitrate.Intendedforcoagulationstudies.

    Selecttheappropriatetubeorsyringesizeforthefishusedinthetrainingsession.Toolargeatubeorsyringecansignificantlyreducethebloodreserveinsmallerfishcausingexcessstressand/ordeath.Paediatrictubesor1mlsyringesshouldbeusedwhenbloodsamplingsmallerfish.

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    Instructtraineesinthepropermethodtoattachanddetachneedlestosyringesorvacutainertubes.

    Stresstheimportanceoffeelingcomfortablewiththebleedingapparatuspriortoinsertionintoalivingfish.

    Traineesshouldpracticemovingtheplungerupanddowninthesyringe.Agoodpracticetoolishavingthetraineesinsertasyringewithattachedneedleintoanorangeandextractasmallamountofjuice.

    Traineesshouldpracticeinsertingavacutainerintothevacutainerholder(withattachedneedle)withoutbreakingthevacuumseal.Abreakinthevacuumsealinairmakesthetubeuselessforbloodcollection,asthebloodwillnotaspirateonceintheveinoraortaofthefish.

    Note:ThefollowingproceduresandphotographshavebeenadaptedwithpermissionoftheauthorfromChapter4.AsepticBacterialExaminationofFinfishin:FinfishandShellfishBacteriologyManualTechniquesandProcedures,2004.WhitmanK.A.(author)BlackwellPublishing,IowaStatePress,Iowa,USA.

    4.6.3.2.TailAblation:

    Fishthataretoosmalltobleedwithasyringeandneedleoravacutainersystemmaynecessitatelethalsamplingbytailablation.

    Euthanizeafish.RefertotheEuthanasiaTemplateforacceptedmethodsofeuthanasia.

    Bloodcollectionfrom Carassiusauratus.(PhotobyR.Hebb)

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    Severthecaudalpedunclewithascalpelbladeorsharpknife.

    Fillahematocrittubewiththebloodasitflowsfromthecaudalvein.

    Plugoneendofthehaematocrittubewithcritoseal.

    DisposeofthefishcarcassandwasteasoutlinedinfacilitySOP.

    4.6.3.3 CaudalVenousPuncture:

    (a)BloodcollectionfromanAmericanshad(Alosasapidissima)usingthecaudalvein(ventralview).(PhotobyR.Hebb)

    (b)Lateral view.(PhotobyR.Hebb)

    Insertaneedleattachedtoasyringeoravacutainersystemundertheskinoftheventralmidlineofthecaudalpeduncleofananaesthetizedorfreshlyeuthanizedfish.

    Alternatively,alateralapproachcanbeusedbyinsertingtheneedleunderthescalesofthemidportionofthetailjustbelow

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    the

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