Background:ForWash-orNo-WashLysingProcedureswithWholeBloodorMarrowSamplesNM-LYSEisapremixed,readytouselysingsolutionfomulatedforlysingerythrocytesfollowingmonoclonalantibodystainingofwholeblood.Treatmentwiththisreagentsimultaneouslyleadstolysisofredbloodcellsandfixationofwhitecells.Morphologicalscattercharacteristicsofleukocytesremainintact.NM-LYSEcanbeusedwithorwithoutsamplewashing.NM-LYSEissuitablefortheanalysisofnormalandmalignantleukocytepopulationsderivedfromvarioushumanBIOLOGicalsamples(blood,bonemarrowandothers)usingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.Flowcytometricanalyseswithmonoclonalantibodiesweresofarrestrictedtoleukocytepopulations,whichhadbeenseparatedfromerythrocytesbeforestainingand/oranalysis.Instead,wholebloodstainingmethodsallowforarapidandaccuratedeterminationofcellularsubpopulationsinnon-separatedbiologicalsamples.ThisisnotonlytimesavingbutreducesalsotheprobABIlityofanunintendedlossofdistinctcellularpopulationsduetoe.g.commonlyuseddifferentialcentrifugationprocedures.WiththeNM-LYSEreagentflowcytometricanalysisofwholebloodhasbecomeaseasyandaccurateastheanalysisofseparatedcellpopulations.Applications:No-WashStainingandLysingProcedure-Foreachsampleadd50µlofEDTAanti-coagulatedbloodtoa3-5mltube-Add20µloftheappropriateNordic-Mubiomonoclonalantibodyconjugate-Incubatethetubefor15minutesat4°Coratroomtemperatureinthedark-Add100µlNM-LYSEtoeachtubeandincubatefor10minutesatroomtemperature-Add1mlofdestilledwaterandvortex,incubatefor5-10minutesatroomtemperature-Analyzeimmediatelyorstoresamplesat2-8°Cinthedarkandanalyzewithin24hoursWashStainingandLysingProcedure-Foreachsampleadd50µlofEDTAanti-coagulatedbloodtoa3-5mltube-Add20µloftheappropriateNordic-MUbiomonoclonalantibodyconjugate-Incubatethetubefor15minutesat4°Coratroomtemperatureinthedark-Add100µlNM-LYSEtoeachtubeandincubatefor10minutesatroomtemperature-Add3-4mlofdestilledwaterandvortex,incubatefor5-10minutesatroomtemperature-Centrifugetubefor5minutesat300g-AspiratesupernatantandresUSPendpelletin0.3mlofsheathfluid-Analyzeimmediatelyorstoresamplesat2-8°Cinthedarkandanalyzewithin24hoursSpecificity:NM-LYSEReagentisdesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufacturer´sinstructionsThequalityofeachNM-LYSELotisdeterminedbylysingredbloodcellsofwelldefinedbloodsamplesfromrepresentativedonorsandsubsequentcomparisonofforwardandsidescattercharacteristicsofobtainedleukocytes.Samples:Biologicalfluids(blood,bonemarrow,andothers)mustbecollectedundersterileconditions.AnticoagulationwithEDTAorheparinisrecommended.Thesamplesshouldbestoredatroomtemperatureuntilused.Foroptimalresults,samplesshouldbeprocessedandanalyzedwithin24hours.Sampleswithhighnumbersofnon-viablecellsmightcausefalseresults,suchcasesrequiredeterminationofcellviabilitywithe.g.propidiumiodide.Allbiologicalsampleshavetobehandledwithcaution.Alwaysconsiderthemaspotentiallyinfective.Useappropriateprecautionssuchasgloves,lab-coat,etc.Limitationsofthetechnique:Flowcytometryshouldbeperformedbyprofessionalusersonly.Improperalignmentoftheflowcytometer,inaccuratecompensationoffluorescenceleakingintootherchannelsaswellasincorrectpositioningofregionsmayleadtofalseresults.LysisofredcellsmightbeimpossIBLeforvariousreasons.Insuchinstancesitisrecommendedtoisolatemononuclearcells(MNC)viadensitygrADIentcentrifugationpriortostaining.Resultswillbecorrectandreproducibleaslongastheproceduresusedrespectthetechnicalrecommendationsandobeygoodlaboratorypractice.TheNM-LYSEsolutionisprovidedataconcentrationthatwillallowlysehumanerythrocytes.Itisthereforestronglyrecommendedtosticktotheworkingprotocolintermsofconcentrationandvolumeregardingcellsandantibody.ThepropertiesofNM-LYSEhavebeendeterminedusingEDTAanti-coagulatedperipheralblood.Avoidingestionandinhalationandcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.Warranty:Theproductssoldhereunderarewarrantedonlytoconformtothequantityandcontentsstatedonthelabelatthetimeofdeliverytothecustomer.Therearenowarranties,expressedorimplied,thatextendbeyondthedescriptiononthelabeloftheproduct.Nordic-MUbio`ssoleliabilityislimitedtoeitherreplacementoftheproductsorrefundofthepurchaseprice.Nordic-MUbioisnotliableforpropertydamage,personalinjury,oreconomiclosscausedbytheproduct.FlowCytometricAnalysis:NM-LYSEReagentisdesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufacturer´sinstructions.Storage:NM-LYSEreagentshouldbestoredandusedatroomtemperature.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.Donotusereagentifaprecipitateshouldformordiscolorationoccurs.Ifunexpectedresultsareobtainedwhichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactus.References:1.Bossuyt,X.,Marti,G.E.&Fleisher,T.A.(1997)Cytometry30,124-33.2.Fritsch,G.,Printz,D.,Stimpfl,M.,Dworzak,M.N.,Witt,V.,Potschger,U.&Buchinger,P.(1997)Transfusion37,775-84.3.Kormoczi,G.F.,Wolfel,U.M.,Rosenkranz,A.R.,Horl,W.H.,Oberbauer,R.&Zlabinger,G.J.(2001)JImmunol167,451-60.4.Menendez,P.,Redondo,O.,Rodriguez,A.,Lopez-Berges,M.C.,Ercilla,G.,Lopez,A.,Duran,A.,Almeida,J.,Perez-Simon,J.A.,SanMiguel,J.F.,Gratama,J.W.&Orfao,A.(1998)Cytometry34,264-71.Caution:Forprofessionalusersonly.NM-LYSEcontainsfomaldehyde.Formaldehydeistoxic,allergenicandasuspectedcarcinogen.Avoidcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.