Background:Flowcytometricanalyseswithmonoclonalantibodiesweresofarrestrictedtoleukocytepopulations,whichhadbeenseparatedfromerythrocytesbeforestainingand/oranalysis.Instead,wholebloodstainingmethodsallowforarapidandaccuratedeterminationofcellularsubpopulationsinnon-separatedBIOLOGicalsamples.ThisisnotonlytimesavingbutreducesalsotheprobABIlityofanunintendedlossofdistinctcellularpopulationsduetoe.g.commonlyuseddifferentialcentrifugationprocedures.WiththeNM-LYSEreagentflowcytometricanalysisofwholebloodhasbecomeaseasyandaccurateastheanalysisofseparatedcellpopulations.Product:NM-LYSEcanbeappliedinwash-orno-washlysingprocedureswithwholebloodorbonemarrowsamples.NM-LYSEisapremixed,readytouselysingsolutionfomulatedforlysingerythrocytesfollowingmonoclonalantibodystainingofwholeblood.Treatmentwiththisreagentsimultaneouslyleadstolysisofredbloodcellsandfixationofwhitecells.Morphologicalscattercharacteristicsofleukocytesremainintact.NM-LYSEcanbeusedwithorwithoutsamplewashing.NM-LYSEissuitablefortheanalysisofnormalandmalignantleukocytepopulationsderivedfromvarioushumanbiologicalsamples(blood,bonemarrowandothers)usingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.ThequalityofeachNM-LYSELotisdeterminedbylysingredbloodcellsofwelldefinedbloodsamplesfromrepresentativedonorsandsubsequentcomparisonofforwardandsidescattercharacteristicsofobtainedleukocytes.Applications:Biologicalfluids(blood,bonemarrow,andothers)mustbecollectedundersterileconditions.AnticoagulationwithEDTAorheparinisrecommended.Thesamplesshouldbestoredatroomtemperatureuntilused.Foroptimalresults,samplesshouldbeprocessedandanalyzedwithin24hours.Sampleswithhighnumbersofnon-viablecellsmightcausefalseresults,suchcasesrequiredeterminationofcellviabilitywithe.g.propidiumiodide.Allbiologicalsampleshavetobehandledwithcaution.Alwaysconsiderthemaspotentiallyinfective.Useappropriateprecautionssuchasgloves,lab-coat,etc.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°Cinthedarkandanalyzewithin24hoursNM-LYSEisdesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufacturer´sinstructions.Limitationsofthetechnique:Flowcytometryshouldbeperformedbyprofessionalusersonly.Improperalignmentoftheflowcytometer,inaccuratecompensationoffluorescenceleakingintootherchannelsaswellasincorrectpositioningofregionsmayleadtofalseresults.LysisofredcellsmightbeimpossIBLeforvariousreasons.Insuchinstancesitisrecommendedtoisolatemononuclearcells(MNC)viadensitygrADIentcentrifugationpriortostaining.Resultswillbecorrectandreproducibleaslongastheproceduresusedrespectthetechnicalrecommendationsandobeygoodlaboratorypractice.TheNM-LYSEsolutionisprovidedataconcentrationthatwillallowlysehumanerythrocytes.Itisthereforestronglyrecommendedtosticktotheworkingprotocolintermsofconcentrationandvolumeregardingcellsandantibody.ThepropertiesofNM-LYSEhavebeendeterminedusingEDTAanti-coagulatedperipheralblood.Avoidingestionandinhalationandcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.Forresearchuseonly.Notfordiagnosticortherapeuticuse.Warranty:Theproductssoldhereunderarewarrantedonlytoconformtothequantityandcontentsstatedonthelabelatthetimeofdeliverytothecustomer.Therearenowarranties,expressedorimplied,thatextendbeyondthedescriptiononthelabeloftheproduct.Nordic-MUbio`ssoleliabilityislimitedtoeitherreplacementoftheproductsorrefundofthepurchaseprice.Nordic-MUbioisnotliableforpropertydamage,personalinjury,oreconomiclosscausedbytheproduct.Storage:NM-LYSEreagentshouldbestoredandusedatroomtemperature.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.Donotusereagentifaprecipitateshouldformordiscolorationoccurs.Ifunexpectedresultsareobtainedwhichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactusReferences: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-LYScontainsfomaldehydeandislabelled:Harmful.Formaldehydeistoxic,allergenicandasuspectedcarcinogen.NeverPipettebymouthandavoidcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.Asamainrule,personsunder18yearsofagearenotallowedtoworkwiththisproduct.Usersmustbecarefullyinstructedintheproperworkingprocedure,thedangerouspropertiesoftheproductandthenecessarysafetyinstructions.PleaserefertotheMaterialSafetyDataSheet(MSDS)foradditionalinformation.Disposeproductremaindersaccordingtolocalregulations.Forresearchuseonly.Notfordiagnosticortherapeuticuse