Beckman Coulter VersaLyse Lysing Solution Ready-for-use User manual

B59380–AD
MADEINFrance
VersaLyse
LysingSolution
.
1mL/test,A09777
100tests;100mL
TABLEOFCONTENTS
English........................................................2
Français(FR).....................................................7
Deutsch(DE)....................................................13
Italiano(IT).....................................................19
Español(ES).....................................................25
PortuguêsPortugal(PT-PT)..............................................31
Dansk(DA).....................................................37
Svenska(SV)....................................................42
Norsk(NO).....................................................47
Ελληνικά(EL)....................................................52
Lietuviškai(LT)....................................................58
Magyar(HU).....................................................63
Polski(PL)......................................................68
Čeština(CZ).....................................................74
Slovenčina(SK)...................................................80
Türkçe(TR).....................................................85
Русский(RU)....................................................90
eestikeel(ET)....................................................96
Hrvatski(HR).....................................................101
Български(BG)...................................................106
Română(RO)....................................................112
Србија(SR).....................................................117
Latviešu(LV).....................................................122
Українська(UK)...................................................127
APPENDIX.....................................................133
REFERENCES....................................................134
©2019BeckmanCoulter,Inc.Allrightsreserved.

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Specications
VersaLyseLysingSolutionReady-for-use
FormulationLiquid
Volume100mL
Numberofvials1vial
Volumepertest1mL(ready-for-usewhenusedwithnoconcomitantxation)
VersaLyse
LysingSolution
.
1mL/test,A09777
100tests;100mL
ForInVitroDiagnosticUse
INTENDEDUSE
Thisreagentisintendedforthelysisofredbloodcellsinthepreparationofbiologicalsamplesforowcytometry
analysis.VersaLyseiscompatiblewithalltypesofcytometersandisusedwithorwithoutwashingafterincubation,
aslongasproperlycalibrateduorescentantibodiesareusedintheprotocol.VersaLysealsomakesitpossibleto
treatsamplesevenwhentheyhavebeenwashedearlier,inordertoeliminateserumimmunoglobulinsforexample
(1,2,3,4,5).
PRINCIPLE
ThesamplecontainingtheredbloodcellstobelysedisexposedtotheVersaLysereagentforatleast10minutes.
ThemajoractiveingredientofVersaLyseisacyclicaminewhich,incontactwithcarbonicanhydrasepresentin
redbloodcells,istransformedintoacompoundwhichishighlylyticforthesecells.
Thisreagentisusedwithspecicconjugatedantibodies,abletobindtoleucocytesduetotheantigenic
determinantstheyexpress.Thespecicstainingofleucocytesisachievedbyincubatingthesamplewiththe
antibodyorantibodies.Theredcellsarethenremovedbylysisandtheleucocytes,unaffectedbythisprocess,
areanalyzedbyowcytometry.
Preparationsmustbestoredbetween2and8°Candanalyzedrapidlyusingowcytometry.Inthecaseofadelay
ofseveralhoursbeforeanalysis,theymaybexedwithPBScontaining0.1%formaldehyde(seethetechnical
leaetoftheIOTest3FixativeSolution–Ref.A07800–topreparethisPBSwith0.1%formaldehyde).Inthis
case,priorwashingisnecessary.
Theowcytometermeasureslightdiffusionandtheuorescenceofcells.Itmakespossiblethedelimitationofthe
populationofinterestwithintheelectronicwindowdenedonahistogram,whichcorrelatestheorthogonaldiffusion
oflight(SideScatterorSS)andthediffusionofnarrow-anglelight(ForwardScatterorFS).Otherhistograms
combiningtwoofthedifferentparametersavailableonthecytometercanbeusedasanaidinthegatingstage
dependingontheapplicationchosenbytheuser.
Theuorescenceofthedelimitedcellsisanalyzedinordertodistinguishthepositively-stainedeventsfromthe
unstainedones.Theresultsareexpressedasapercentageofpositiveeventsinrelationtoalltheeventsacquired
bythegating.
WARNINGANDPRECAUTIONS
1.Donotusethereagentbeyondtheexpirydate.
2.Donotstoreintherefrigerator,donotfreeze.
3.Minimizeexposuretolightduringincubationwithuorescentantibodies.
4.Avoidmicrobialcontaminationofthereagents,orfalseresultsmayoccur.
5.Formaldehydeistoxicandallergenic.Itisthoughttobecarcinogenic.Neverpipettebymouthandavoidall
contactwiththeskin,mucosa,eyesandclothing.
6.Allbloodsamplesmustbeconsideredaspotentiallyinfectiousandmustbehandledwithcare(inparticular:
thewearingofprotectivegloves,gownsandgoggles).
7.Bloodtubesanddisposablematerialusedforhandlingshouldbedisposedofinadhoccontainersintended
forincineration.

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GHSHAZARDCLASSIFICATION
VersaLyseLysingSolutionWARNING
H315Causesskinirritation.
H319Causesseriouseyeirritation.
P280Wearprotectivegloves,protective
clothingandeye/faceprotection.
P302+P352IFONSKIN:Washwithplentyof
soapandwater.
P305+P351+P338IFINEYES:Rinsecautiouslywith
waterforseveralminutes.Remove
contactlenses,ifpresentandeasy
todo.Continuerinsing.
P332+P313Ifskinirritationoccurs:Getmedical
advice/attention.
P337+P313Ifeyeirritationpersists:Get
medicaladvice/attention.
P362+P364Takeoffcontaminatedclothingand
washitbeforeuse.
HydrochloricAcid0.5-1.0%
Pyrrolidine1-2%
SafetyDataSheetisavailableattechdocs.beckmancoulter.com
STORAGEANDSTABILITY
VersaLyseisstoredbetween18and25°C.IfavialofVersaLysehasbeenkeptinadvertantlyinarefrigerator,bring
ittoroomtemperature(18–25°C)andwaitatleastonehourbeforeuse.
Withthevialclosed,thereagentisstableuptotheexpirydateshownonthevial.
Afteropening,thereagentisstablefor90days.
SAMPLES
VenousbloodorbonemarrowsamplesmustbetakenusingsteriletubescontaininganEDTAsaltasthe
anticoagulant.
NB:CarefullyreadintheANTICOAGULANTsection,theconditionsunderwhichotheranticoagulantscanbeused.
Thesamplesshouldbekeptatroomtemperature(18–25°C)andnotshaken.Thesampleshouldbehomogenized
bygentleagitationpriortotakingthetestsample.
Thesamplesmustbeanalyzedwithin24hoursofvenipuncture.
WASHEDSAMPLES
Ifyouhavetowashthesamplesbeforestainingfollowedbylysisoftheredbloodcells,itisessentialtoremove
allthesupernatantafterthelastcentrifugation.Anexcessofwashingbufferonthecellpelletcanreducethe
effectivenessofVersaLyse.
PREPARATIONOFSAMPLES
Theconcentrationofleucocytesinthesamplemustbelessthan104cells/µL(1010/L).Ifnecessary,diluteinPBS
tobringtheleucocyteconcentrationto5x103/µL(5x109/L).
Theerythrocyticconcentrationinthesamplemustbelessthan6x106/µL(6x1012/L),itisrecommendedtodilute
sampleinPBSinordertobringtheerythrocyticconcentrationto5x106/µL.
Ifboththeleucocyticanderythrocyticconcentrationsneedadjusting,thehighestdilutionmustbeused.The
proceduresuse100µLofanon-predilutedorpre-dilutedsamplepertube.
EVIDENCEOFDETERIORATION
Anychangeinthephysicalappearanceofthereagentsmayindicatedeteriorationandthereagentshouldnotbe
used.

B59380–AD4of135
Incaseofpackagingdeteriorationorifdataobtainedshowsomeperformancealteration,pleasecontactyourlocal
distributororusethefollowinge-mailaddress:
[e-mail]
MATERIALSNEEDEDBUTNOTSUPPLIEDWITHKIT:
•Samplingtubesandmaterialnecessaryforsampling.
•Automaticpipetteswithdisposabletipsfor10,20,100and1,000µL.
•Plastichaemolysistubes.
•Calibrationbeads:Flow-SetFluorospheres(Ref.6607007).
•Specicuorescentantibodies.
•Isotypiccontrols.
•Buffer(PBS:0.01Msodiumphosphate;0.145Msodiumchloride;pH7.2).
•Fixationreagent:forexampleIOTest3FixativeSolution(Ref.A07800).
•Centrifuge.
•Automaticagitator(Vortextype).
•Flowcytometer.
PROCEDUREwithVERSALYSEREAGENT
Note:Theproceduresbelowarevalidforstandardapplications.Sampleand/orVersaLysevolumesforcertain
BeckmanCoulterapplicationsmaybedifferent.Ifsuchisthecase,followtheinstructionsonthetechnicalleaet
oftheapplication.
A–Procedurewithoutconcomitantxation
Preparationofthereagent
Nopreparationisnecessary.UseVersaLysedirectlyfromthevial.
Foreachsampleanalyzed,inadditiontothetesttube,onecontroltubeisrequiredinwhichtheisotypiccontrol
correspondingtothespecicstainingselectedisaddedtothecells.
1.Ineachtesttubeaddtheamountofantibodyrecommendedbythemanufacturerinordertostain5x105
leucocytes.
2.Ineachcontroltubeaddtheamountoftheisotypiccontrolrecommendedbythemanufacturertostain5x105
leucocytes.
3.Add100µLofthesample(predilutedorotherwise)tobothtubes.Vortexthetubesgently.
4.Incubatefollowingtheconditionssetoutinthetechnicalleaetfortheantibodiesused.
5.Add1mLofVersaLyseandvorteximmediatelyfor1second.
6.Incubateatleast10minutesatroomtemperature(18–25°C),protectedfromlight.
Inthecaseofaprocedurewithoutwashing,thetubesarereadyforcytometricanalysis.
NB:Ifthepreparationsneedtobewashed,donotanalyzebeforeperformingsteps7to11.
7.Centrifugefor5minutesat150xgatroomtemperature.
IMPORTANT:Ithasbeennotedthat,whenusingthenonconcomitantxationprocedurewithawashstep,
celldoublets,knownasescapees,maybeformed,possiblyresultinginerroneousresultsduetochangein
theirlightscatterpropertiesandtothefactthattheyescapeoutofthegatingregions(6).Arigorousvortexing
maydisruptthesedoublets.
8.Removethesupernatantbyaspiration.
9.Resuspendthecellpelletin3mLofPBS.
10.Centrifugefor5minutesat150xgatroomtemperature.
11.Removethesupernatantbyaspirationandresuspendthecellpelletin:
•0.5mLor1mLofPBSplus0.1%offormaldehydeifthepreparationsaretobekeptlessthan24hours.(A
0.1%formaldehydePBScanbeobtainedbydiluting12.5µLoftheIOTest3FixativeSolution(Ref.A07800)
atits10Xconcentrationin1mLofPBS).
•0.5mLor1mLofPBSwithoutformaldehyde,ifthepreparationsaretobeanalyzedwithin2hours.

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Note:Inallcases,keepthepreparationsatbetween2and8°Candprotectedfromlight.
B–Procedurewithconcomitantxation
Preparationofthereagent
Extemporaneouslypreparethe“Fix-and-Lyse”mixturebyadding25µLofUNDILUTEDIOTest310X*Fixative
Solution(Ref.A07800)to1mLofVersaLyse.
Vortexthetubesfor3to5seconds.
(*)NB:inthecontextofthe“Fix-and-Lyse“mixture,theIOTest3Fixativesolution(Ref.A07800)isusedataforty
foldconcentratedsolutionandnotasa10Xsolution(nominalconcentration).
Prepareasufcientvolumeofthe“Fix-andLyse”mixturedependingonthenumberofsamplestobelysed(1mL
ofmixturepertube).
Foreachsampleanalyzed,inadditiontothetesttube,onecontroltubeisrequiredinwhichthecellsaremixed
withtheisotypiccontrolcorrespondingtothespecicstainingselected.
1.Ineachtesttube,addtheamountofantibodyrecommendedbythemanufacturertostain5x105leucocytes.
2.Ineachcontroltube,addtheamountofisotypiccontrolrecommendedbythemanufacturertostain5x105
leucocytes.
3.Add100µLofthesample(predilutedorotherwise)tobothtubes.Vortexthetubesgently.
4.Incubatefollowingtheconditionssetoutinthetechnicalleaetfortheantibodiesused.
5.Add1mLofthe“Fix-and-Lyse“mixtureandvorteximmediatelyfor1second.
6.Incubateatleast10minutesatroomtemperature(18–25°C),protectedfromlight.
Inthecaseofaprocedurewithoutwashing,thetubesarereadyforcytometricanalysis.
NB:Ifthepreparationsneedtobewashed,donotanalyzebeforeperformingsteps7to11.
7.Centrifugefor5minutesat150xgatroomtemperature.
8.Removethesupernatantbyaspiration.
9.Resuspendthecellpelletin3mLofPBS.
10.Centrifugefor5minutesat150xgatroomtemperature.
11.Removethesupernatantbyaspirationandresuspendthecellpelletin0.5mLor1mLofPBSplus0.1%
offormaldehydeifthepreparationsaretobekeptlessthan24hours.(A0.1%formaldehydePBScanbe
obtainedbydiluting12.5µLoftheIOTest3FixativeSolution(Ref.A07800)atits10Xconcentrationin1mL
ofPBS).
Thesepreparationsmaybekept24hoursbetween2and8°Candprotectedfromlight.
ANTICOAGULANT
ThetripotassiumsaltofEDTA(K3EDTA)givesthebestresults,irrespectiveofthemethodologyused,withor
withoutwashingafterlysis.
ACDandheparingivegoodresultswithallproceduresexcepttheprocedurewithoutconcomitantxationwhenitis
followedbywashing.Withtheseanticoagulants,werecommendtheprocedurewithconcomitantxation,followed
bywashing.
ANALYSIS
Theerythrocyteresiduescangiverisetoadiffractionsignalofthenarrowanglelight(ForwardScatterorFS)
greaterthanthatobtainedwithothercommerciallysisreagents.Asaresult,theusualadjustment(orxedby
default)ofthe("threshold")discriminatoronthisparametermaybetooweakforVersaLyse.
Itisthereforeadvisabletoadjustthecytometerduringtherstmomentsoftheacquisitionofatypicalpreparation.In
thecaseofwholelysedbloodforexample,itisadvisablersttoincreaseprogressivelythevalueofthediscriminator
untiltheleucocytepopulationsarevisibleonahistogramoftheFStypeversusSS;thentocarefullyadjustthe
amplicationoftheFSandSSsignalstoobtainadistributionoftheleucocyticpopulationsidenticaltothatofgures
1and2.
PERFORMANCE
purityandlymphocyticrecovery
PurityandlymphocyticrecoveryhavebeenevaluatedaccordingtotherecommendationsoftheCDC(1).The
bloodof10healthydonorssampledinK3EDTAwaslabeledwithamixtureofmonoclonalantibodiesCD45-FITC