Background:

Flowcytometricanalyseswithmonoclonalantibodiesweresofarmainlyrestrictedtocellsurfacemolecules.Intracellularstructuressuchascytoplasmicornuclearenzymes,oncoproteins,cytokines,immunoglobulinsetc.werelargelyexcludedfromsuchstudies.Alsoexcludedfromflowcytometricstudieswerecytoplasmiclocalizationsofwell-establishedmembranemoleculeslikeCD3andCD22,which,intheircytoplasmicform,arethemostreliablelineageMarkersinundifferentiatedleukemia.WiththeFIX&PERM®Kitflowcytometricanalysisofintracellularantigenshasbecomeaseasyassurfaceantigenstudies.TheonlyprerequisiteistheavailABIlityofsuitableantibodyconjugates.MostoftheavailablemonoclonalantibodyconjugatescanbeusedwiththeFIX&PERM®Kit,somedeterminantsaresensitive,however,tothefixationstepinvolved.Thisandtheoptimalfixationtimehavetobetestedforeachreagent.

Product:

ThisFIX&PERM®CellFixationandPermeabilizationKitcontains2reagents:FixationMedium(ReagentA)andPermeabilizationMedium(ReagentB).ItisintendedforfirstfixingcellsinsUSPensionwithReagentAandthenpermeabilizingthecellmembraneswithReagentB.Thisproceduregivesantibodiesaccesstointracellularstructuresandleavesthemorphologicalscattercharacteristicsofcellsintact.SpecificformulationsreducebackgroundstainingandallowsimultaneousadditionofpermeabilizationmediumandFluorochromelabeledantibodies.FIX&PERM®issuitablefortheanalysisofnormalandmalignantleukocytepopulationsderivedfromvarioushumanBIOLOGicalsamples(blood,bonemarrowandothers)usingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.

Format:1x5mlvialofFixationMedium(ReagentA)and1x5mlvialPermeabilizationMedium(ReagentB).FIX&PERM®Reagentsaredesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufacturer´sinstructions.

ThequalityofeachFIX&PERM®Lotisdeterminedbyfixationandpermeabilizationofwelldefinedbloodsamplesfromrepresentativedonorsandsubsequentcomparisonofforwardandsidescattercharacteristicsofobtainedleukocytes,aswellasimmunostainingefficiencyforseveralmembranousandcytoplasmicantigens

Applications:

Biologicalfluids(blood,bonemarrow,andothers)mustbecollectedundersterileconditions.AnticoagulationwithEDTAorheparinisrecommended.Thesamplesshouldbestoredatroomtemperatureuntilused.Foroptimalresults,samplesshouldbeprocessedandanalyzedwithin24hours.Sampleswithhighnumbersofnon-viablecellsmightcausefalseresults,suchcasesrequiredeterminationofcellviabilitywithe.g.propidiumiodide.Allbiologicalsampleshavetobehandledwithcaution.Alwaysconsiderthemaspotentiallyinfective.Useappropriateprecautionssuchasgloves,lab-coat,etc.

Fixation,permeabilizationandstainingprocedure:

•Foreachsampletobeanalyzedadd50µlofwholeblood,bonemarrowormononuclearcellsuspensionina5mltube
•Add100µlofReagentA(FixationMedium,storedandusedatroomtemperature)
•Incubatefor15minutesatroomtemperature
•Add5mlphosphatebufferedsalineandcentrifugecellsfor5minutesat300g
•Removesupernatantandaddtocellpellet100µlReagentB(PermeabilizationMedium)and20µloftheappropriateNordic-Mubiomonoclonalantibodyconjugate
•Vortexatlowspeedfor1-2seconds
•Incubatefor15minutesatroomtemperature
•Washcellswithphosphatebufferedsalineasdescribedabove
•Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1.0%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hours.


Comments:Specialcases(dilutedbonemarrowsamples,othersamplescontaininglowsolubleprotein)mightbenefitfromreplenishmentwithplasmacomponentsbeforetheFIX&PERM®treatmentinordertocreateamilieu,whichmorecloselyresemblesthestuationinanti-coagulatedblood.ForthatpuposeadditionofIgGpreparations(e.g.BeriglobulinP,ZLBBehring,finalconcentration10mg/ml)andhumanserumalbumin(e.g.humanalbumin“Behring”20%-infusionsolution,finalconcentration40mg/ml)isrecommended.

Limitationsofthetechnique:

Flowcytometryshouldbeperformedbyprofessionalusersonly.Improperalignmentoftheflowcytometer,inaccuratecompensationoffluorescenceleakingintootherchannelsaswellasincorrectpositioningofregionsmayleadtofalseresults.
LysisofredcellsmightbeimpossIBLeforvariousreasons.Insuchinstancesitisrecommendedtoisolatemononuclearcells(MNC)viadensitygrADIentcentrifugationpriortostaining.

Resultswillbecorrectandreproducibleaslongastheproceduresusedrespectthetechnicalrecommendationsandobeygoodlaboratorypractice.

TheFIX&PERM®solutionsareprovidedinaconcentrationthatwillallowtofixandpermeabilizehumanhematopoieticcells.Itisthereforestronglyrecommendedtosticktotheworkingprotocolintermsofconcentrationandvolumeregardingcellsandantibody.ThepropertiesofFIX&PERM®havebeendeterminedusingEDTAanti-coagulatedperipheralblood.

Warranty:

Theproductssoldhereunderarewarrantedonlytoconformtothequantityandcontentsstatedonthelabelatthetimeofdeliverytothecustomer.Therearenowarranties,expressedorimplied,thatextendbeyondthedescriptiononthelabeloftheproduct.Nordic-MUbio`ssoleliabilityislimitedtoeitherreplacementoftheproductsorrefundofthepurchaseprice.Nordic-MUbioisnotliableforpropertydamage,personalinjury,oreconomiclosscausedbytheproduct.

Storage:

FIX&PERM®CellFixationandPermeabilizationKitreagentsshouldbestoredandusedatroomtemperature.Donotfreeze.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.Ifreagentsarestoredunderanyconditionsotherthanthosespecified,theconditionsmustbeverifiedbytheuser.Donotusereagentsifaprecipitateshouldformordiscolorationoccurs.Ifunexpectedresultsareobtainedwhichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactus.

References:

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Caution:

Forprofessionalusersonly.ReagentAofFIX&PERM®CellFixationandPermeabilizationKitcontainsfomaldehydeandislabelled:Harmful.Formaldehydeistoxic,allergenicandasuspectedcarcinogen.NeverPipettebymouthandavoidcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.Asamainrule,personsunder18yearsofagearenotallowedtoworkwiththisproduct.Usersmustbecarefullyinstructedintheproperworkingprocedure,thedangerouspropertiesoftheproductandthenecessarysafetyinstructions.PleaserefertotheMaterialSafetyDataSheet(MSDS)foradditionalinformation.Disposeproductremaindersaccordingtolocalregulations.