Beckman Coulter CD103-FITC User manual

B77267–AC
MADEINFrance
IOT est
ConjugatedAntibody
CD103-FITC
B49222100tests;2mL,20µL/test
TABLEOFCONTENTS
English........................................................2
Français(FR).....................................................5
Deutsch(DE).....................................................8
Italiano(IT).....................................................11
Español(ES).....................................................14
PortuguêsPortugal(PT-PT)..............................................17
Dansk(DA).....................................................20
Svenska(SV)....................................................23
Norsk(NO).....................................................26
Ελληνικά(EL)....................................................29
Lietuviškai(LT)....................................................32
Magyar(HU).....................................................35
Polski(PL)......................................................38
Čeština(CZ).....................................................41
Slovenčina(SK)...................................................44
한국어(KO).....................................................47
Русский(RU)....................................................50
eestikeel(ET)....................................................53
Hrvatski(HR).....................................................56
Български(BG)...................................................59
Română(RO)....................................................62
Србија(SR).....................................................65
Latviešu(LV).....................................................68
Українська(UK)...................................................71
APPENDIX.....................................................74
REFERENCES....................................................75
©2019BeckmanCoulter,Inc.Allrightsreserved.

B77267–AC2of76
Specications
SpecicityCD103
Clone2G5
HybridomaNS1xbalb/c
ImmunogenHumanintestinallymphocytes(HML1)
ImmunoglobulinIgG2a
SpeciesMouse
PuricationAfnitychromatography
FluorochromeFluoresceinisothiocyanate(FITC)
MolarratioFITC/Ig:5.0-7.0
λexcitation488nm
EmissionPeak525nm
BufferPBSpH7.2plus2mg/mLBSAand0.1%NaN3
IOT est
ConjugatedAntibody
CD103-FITC
B49222100tests;2mL,20µL/test
Forinvitrodiagnosticuse.
INTENDEDUSE
Thisuorochrome-conjugatedantibodyallowstheidenticationofcellpopulationsexpressingtheCD103antigenpresentinhumanperipheralbloodusing
owcytometry.
PRINCIPLE
Thistestisbasedontheabilityofspecicmonoclonalantibodiestobindtotheantigenicdeterminantsexpressedbyleucocytes.
SpecicstainingoftheleucocytesisperformedbyincubatingthesamplewiththeIOTestreagent.Theredcellsarethenremovedbylysisandtheleucocytes,
whichareunaffectedbythisprocess,areanalyzedbyowcytometry.
Theowcytometermeasureslightdiffusionandtheuorescenceofcells.Itmakespossiblethedelimitationofthepopulationofinterestwithintheelectronic
windowdenedonahistogram,whichcorrelatestheorthogonaldiffusionoflight(SideScatterorSS)andthediffusionofnarrow-anglelight(ForwardScatter
orFS).Otherhistogramscombiningtwoofthedifferentparametersavailableonthecytometercanbeusedassupportsinthegatingstagedependingonthe
applicationchosenbytheuser.
EXAMPLESOFCLINICALAPPLICATIONS
CD103isexpressedinseveralsubsetsoflymphocytesincludingasmallsubsetofperipherallymphocytes(Tregcells),onactivatedandTGF-betastimulated
lymphocytes,onintestinalintraepitheliallymphocytes,laminaproprialymphocytes,epithelialdendriticcells,andlaminapropria-deriveddendriticcells.CD103⁺
dendriticcellsintheintestinalmucosallaminapropriaappeartoplayanimportantroleinmaintainingintestinalimmunehomeostasisinducingtolerogenic
immuneresponsesandimprintingphenotypicchangesonantigen-specicTcells.
DCsexpressingCD103intheintestineinduceFoxp3T-regsfromFoxp3-CD4TcellswithendogenousTGF-βandretinoicacid(1,2).Also,apopulationof
CD8+CD103+TcellshasbeendescribedwitheffectorandTregulatorycellfunctionsparticipatingasefferentintheallograftrejectionandsuppressorof
cytotoxicity/proliferation,respectively(3,4).CD103isincludedinthepaneldistinctivefortheimmunophenotypiccharacterizationofHairycellleukemiathat
includeabnormallybrightCD20+CD22+,CD11c+,CD25+,positiveCD103+,CD123+associatedwithnegativeCD5-,CD10-andCD23-(5,6,7,8).
REAGENTS
Concentration:SeelotspecicCerticateofAnalysisatwww.beckmancoulter.com.
WARNINGANDPRECAUTIONS
1.Donotusethereagentbeyondtheexpirydate.
2.Donotfreeze.
3.Letitcometoroomtemperature(18–25°C)beforeuse.
4.Minimizeexposuretolight.
5.Avoidmicrobialcontaminationofthereagents,orfalseresultsmayoccur.
6.Antibodysolutionscontainingsodiumazide(NaN3)shouldbehandledwithcare.Donottakeinternallyandavoidallcontactwiththeskin,mucosa
andeyes.
Moreover,inanacidmedium,sodiumazidecanformthepotentiallydangeroushydrazoicacid.Ifitneedstobedisposedof,itisrecommendedthat
thereagentbedilutedinalargevolumeofwaterbeforepouringitintothedrainagesystemsoastoavoidtheaccumulationofsodiumazideinmetal
pipesandtopreventtheriskofexplosion.
7.Allbloodsamplesmustbeconsideredaspotentiallyinfectiousandmustbehandledwithcare(inparticular:thewearingofprotectivegloves,gowns
andgoggles).
8.Neverpipetbymouthandavoidallcontactofthesampleswiththeskin,mucosaandeyes.
9.Bloodtubesanddisposablematerialusedforhandlingshouldbedisposedofinadhoccontainersintendedforincineration.
10.Reagentsandwasteshouldbeeliminatedaccordingtolocalrequirements.
GHSHAZARDCLASSIFICATION
Notclassiedashazardous

B77267–AC3of76
SafetyDataSheetisavailableatbeckman.com/techdocs
STORAGEANDSTABILITY
Theconjugatedliquidformsmustbekeptatbetween2and8°Candprotectedfromlight,beforeandafterthevialhasbeenopened.
Stabilityofclosedvial:seeexpirydateonvial.
Stabilityofopenedvial:thereagentisstablefor180days.
SAMPLES
VenousbloodmustbetakenusingsteriletubescontaininganEDTAsaltastheanticoagulant.
Thesamplesshouldbekeptatroomtemperature(18–25°C)andnotshaken.Thesamplesshouldbehomogenizedbygentleagitationpriortotakingthe
testsample.
Thesamplesmustbeanalyzedwithin24hoursofvenipuncture.
EVIDENCEOFDETERIORATION
Anychangeinthephysicalappearanceofthereagentsmayindicatedeteriorationandthereagentshouldnotbeused.
Incaseofpackagingdeteriorationorifdataobtainedshowsomeperformancealteration,pleasecontactyourlocaldistributororusethefollowinge-mail
address:[e-mail]
CONTENTS
Sodiumazidepreservativemayformexplosivecompoundsinmetaldrainlines.SeeNIOSHBulletin:ExplosiveAzideHazard(8/16/76).
Toavoidthepossiblebuild-upofazidecompounds,ushwastepipeswithwaterafterthedisposalofundilutedreagent.Sodiumazidedisposalmustbein
accordancewithappropriatelocalregulations.
MATERIALSNEEDEDBUTNOTSUPPLIEDWITHKIT:
•Samplingtubesandmaterialnecessaryforsampling.
•Automaticpipetteswithdisposabletipsfor20,100and500µL.
•Plastichaemolysistubes.
•Calibrationbeads:Flow-SetProFluorospheres(Ref.A63492)Flow-CheckProFluorospheres(Ref.A63493)
•Redcelllysisreagentwithwashingstageafterlysis.Forexample:VersaLyse(Ref.A09777).
•Leucocytexationreagent.Forexample:IOTest3FixativeSolution(Ref.A07800).
•Isotypiccontrol:FITCIOTestreagent(Ref.A12689)
•Buffer(PBS:0.01Msodiumphosphate;0.145Msodiumchloride;pH7.2).
•Centrifuge.
•Automaticagitator(Vortextype).
•Flowcytometer.
PROCEDURE
ProcedurewithVersaLysereagent
Foreachsampleanalyzed,inadditiontothetesttube,onecontroltubeisrecommendedinwhichthecellsaremixedinthepresenceoftheisotypiccontrol
(Ref.A12689)
1.Add20µLofspecicIOTestconjugatedantibodytoeachtesttube,and20µLoftheisotypiccontroltoeachcontroltube.
2.Add100µLofthetestsampletobothtubes.Vortexthetubesgently.
3.Incubatefor15to20minutesatroomtemperature(18–25°C),protectedfromlight.
4.Thenperformlysisoftheredcells,byfollowingtherecommendationsofthelysisreagentused.
Forexample,ifyouwishtouseVersaLyse(Ref.A09777),refertotheleaetandfollowpreferablytheprocedurecalled“withconcomitantxation”,
whichconsistsinadding1mLofthe“Fix-and-Lyse”mixturepreparedextemporaneously.Vorteximmediatelyforonesecondandincubatefor10
minutesatroomtemperature,protectedfromlight.
5.Centrifugefor5minutesat150xgatroomtemperature.
6.Removethesupernatantbyaspiration.
7.Resuspendthecellpelletusing3mLofPBS.
8.Repeatstep5.
9.Removethesupernatantbyaspirationandresuspendthecellpelletusing:
0.5mLofPBSplus0.1%offormaldehydeifthepreparationsaretobekeptlessthan24hours.(A0.1%formaldehydePBScanbeobtainedbydiluting
12.5µLoftheIOTest3FixativeSolution(Ref.A07800)atits10Xconcentrationin1mLofPBS).
0.5mLofPBSwithoutformaldehyde,ifthepreparationsaretobeanalyzedwithin2hours.
Note:Inallcases,keepthepreparationsbetween2and8°Candprotectedfromlight.
PERFORMANCE
Performancedataareobtainedusingtheproceduredescribedaboveonsamplesspikedwithcelllinewithin24hoursofvenipuncturepreviouslycollectedon
steriletubeswithEDTAsaltasanticoagulantandstoredat18-25°C.Analysisisperformedwithin24hoursfollowingimmunostaining.
SPECIFICITY
CD103(αEβ7)belongstotheintegrinfamilymolecules.ItisaheterodimerconsistingofanαEsubunitandaβ7subunit.CD103isinmatureformcleaved
intotwodisulde-linkedchains(C-terminal150kDachainandN-terminal25kDachain).TheheterodimerizationofCD103withintegrinbeta7subunitleadto

B77267–AC4of76
theformationofalphaE/beta7integrin(mucosallymphocyte1antigen).ThecomplexαEβ7isaligandforE-cadherin(whichismainlyexpressedinepithelial
cells)andaninteractionbetweenαEβ7andE-cadherinresultsinadhesionoflymphocytestoepithelialcells.CD103bindingtoE-cadherinmediateshomingof
lymphocytestotheintestinalepithelium,and,inadditiontotheroleinadhesion,mayserveasanaccessorymoleculeforintraepitheliallymphocyteactivation.
The2G5monoclonalantibody(mAb)stainsallthevarioussubsetsofhumanintraepitheliallymphocytes(IEL)aswellassubpopulationsoflaminapropriaT
cellsandmesentericlymphoblasts.Itreactsalsowithsomelymphocytesinothermucosae(9,10).
The2G5mAbhasbeenassignedtotheCD103clusterofdifferentiationduringthe5thInternationalWorkshoponHumanLeucocyteDifferentiationAntigens,
inBoston,USA,in1993(WSCode:A005)(11).
LIMITOFDETECTION
LIMITOFDETECTIONWITHVERSALYSE
AstudywasconductedinaccordancewithCLSIEP17-A2,EvaluationofDetectionCapabilityforClinicalLaboratoryMeasurementProcedures;Approved
Guideline-secondEdition(12).TheLimitofDetection(LOD)isthelowestconcentrationofanalytethatcanbeconsistentlydetected.Resultsobtainedare
summarizedinthefollowingtable:
PositiveTargetLimitofDetection
Molt16+CD103-FITC+1.0cell/μL
PRECISION
PRECISIONWITHVERSALYSE
Thepercentageofstainingofapositivetargetwasdeterminedusingtwowholebloodsamples,runintenreplicates,onthesamedayandusingthesame
cytometer.
Theresultsobtainedaresummarizedinthefollowingtable:
Mean(%)SDCV(%)
PositiveTargetNumber
Donor1Donor2Donor1Donor2Donor1Donor2
Molt16+CD103-FITC+10100.00100.000.010.010.010.01
LIMITATIONS
1.Flowcytometrymayproducefalseresultsifthecytometerhasnotbeenalignedperfectly,ifuorescenceleakshavenotbeencorrectlycompensated
forandiftheregionshavenotbeencarefullypositioned.
2.ItispreferabletouseaRBClysistechniquewithawashingstepasthisreagenthasnotbeenoptimizedfor"nowash"lysistechniques.
3.Accurateandreproducibleresultswillbeobtainedaslongastheproceduresusedareinaccordancewiththetechnicalinsertleaetandcompatible
withgoodlaboratorypractices.
4.Theconjugatedantibodyofthisreagentiscalibratedsoastoofferthebestspecicsignal/non-specicsignalratio.Therefore,itisimportanttoadhere
tothereagentvolume/samplevolumeratioineverytest.
5.Inthecaseofahyperleucocytosis,dilutethebloodinPBSsoastoobtainavalueofapproximately5x109leucocytes/L(13).
6.Incertaindiseasestates,suchassevererenalfailureorhaemoglobinopathies,lysisofredcellsmaybeslow,incompleteorevenimpossible.Inthis
case,itisrecommendedtoisolatemononucleatedcellsusingadensitygradient(Ficoll,forexample)priortostaining(14).
TRADEMARKS
BeckmanCoulter,thestylizedlogo,andtheBeckmanCoulterproductandservicemarksmentionedhereinaretrademarksorregisteredtrademarksof
BeckmanCoulter,Inc.intheUnitedStatesandothercountries.
SymbolsKey
GlossaryofSymbolsisavailableatbeckman.com/techdocs(documentnumberB60062)

B77267–AC5of76
Spécications
SpécicitéCD103
Clone2G5
HybridomeNS1xbalb/c
ImmunogèneHumanintestinallymphocytes(HML1)
ImmunoglobulineIgG2a
EspècesSouris
PuricationChromatographied'afnité
FluorochromeFluoresceinisothiocyanate(FITC)
RatioMolaireFITC/Ig:5,0-7,0
λd’excitation488nm
Picd’émission525nm
TamponPBSpH7,2additionnéde2mg/mLBSAetde0,1%NaN3
IOT est
ConjugatedAntibody
CD103-FITC
B49222100tests;2mL,20µL/test
Pourunusagediagnostiqueinvitro.
UTILISATION
Cetanticorpsconjuguéauuorochromepermetl'identicationdespopulationsdecellulesexprimantl'antigèneCD103présentdanslesangpériphérique
humainenutilisantlacytométriedeux.
PRINCIPE
Cetestreposesurlacapacitéd’anticorpsmonoclonauxspéciquesdesexersurdescellulesleucocytairesparlesdéterminantsantigéniquesqu’elles
expriment.
Lemarquagespéciquedesleucocytesestréaliséenincubantl’échantillonavecleréactifIOT est.Lesérythrocytessontensuiteéliminésparlyseetles
leucocytes,nonaffectésparcetteétape,sontanalysésparcytométrieenux.
Lecytomètredeuxmesureladiffusiondelalumièreainsiquelauorescencedescellules.Ilpermetladélimitationdelapopulationd’intérêtàl'intérieur
d’unefenêtreélectronique,déniesurunhistogrammequicorrèleladiffusionorthogonaledelalumière("SideScatter"ouSS)etladiffusiondelalumière
auxanglesétroits(“ForwardScatter”ouFS).D’autreshistogrammescombinantdeuxdesdifférentsparamètresdisponiblessurlecytomètresontutilisables
commesupportsàl’étapedegatingélectroniqueenfonctiondel’applicationchoisieparl’utilisateur.
EXEMPLESD'APPLICATIONSCLINIQUES
LeCD103estexprimédansplusieurssous-ensemblesdelymphocytes,parmilesquelsunpetitsous-ensembledelymphocytespériphériques(lymphocytes
Trégulateurs),surleslymphocytesactivésetstimulésparTGF-bêta,surleslymphocytesintraépithéliauxintestinaux,leslymphocyteslaminapropria,
lescellulesdendritiquesépithélialesetlescellulesdendritiquesdérivéesdelalaminapropria.LescellulesdendritiquesCD103⁺delalaminapropriade
lamuqueuseintestinalesemblentjouerunrôleimportantdanslemaintiendel’homéostaseimmunitaireintestinaleinduisantdesréponsesimmunitaires
tolérogéniquesetimprimantdesmodicationsdephénotypesurleslymphocytesTspéciquesauxantigènes.
LesDCexprimantleCD103dansl’intestininduisentleslymphocytesTrégulateursFoxp3extraitsdeslymphocytesTFoxp3-CD4avecduTGF-βetdel’acide
rétinoïque(1,2).Enoutre,unepopulationdelymphocytesTCD8+CD103+aétédécriteaveclesfonctionsdeslymphocyteseffecteursetdeslymphocytes
Trégulateursparticipantentantqu’efférentsdanslerejetdel’allogreffeetentantquesuppresseursdelacytotoxicité/prolifération,respectivement(3,4).
LeCD103estinclusdanslepaneldistinctifpermettantlacaractérisationimmunophénotypiquedelaleucémieàtricholeucocytesquiinclutlescellules
anormalementlumineusesCD20+CD22+,CD11c+,CD25+,positivesCD103+,CD123+associéesàdescellulesnégativesCD5-,CD10-etCD23-(5,6,7,8).
RÉACTIFS
Concentration:voirlecerticatd'analysespéciquedulotsurwww.beckmancoulter.com.
AVERTISSEMENTSETPRÉCAUTIONS
1.Nepasutiliserleréactifau-delàdeladatedepéremption.
2.Nepascongeler.
3.Laisserreveniràtempératureambiante(18–25°C)avantutilisation.
4.Minimiserlestempsd’expositionàlalumière.
5.Eviterlacontaminationmicrobiennedesréactifsoudesrésultatserronéspeuventapparaître.
6.Lessolutionsd'anticorpscontenantdel'azoturedesodium(NaN3)sontàmanipuleravecprécaution.Nepasavaleretévitertoutcontactaveclapeau,
lesmuqueusesetlesyeux.
Parailleurs,enmilieuacide,l'azoturedesodiumpeutformerdel'acidehydrazoïquepotentiellementdangereux.Lorsdel'élimination,ilestrecommandé
dediluerleréactifdansungrandvolumed'eauavantdeleverserdanslesystèmed'évacuationdeseauxpouréviterl'accumulationd'azoturedesodium
danslescanalisationsmétalliquesetprévenirlerisqued'explosion.
7.Toutéchantillonsanguindoitêtreconsidérécommepotentiellementinfectieuxetdoitêtremanipuléaveclesprécautionsd'usage(enparticulier:port
degants,deblouseetdelunettesdeprotection).
8.Nejamaispipeteràlaboucheetévitertoutcontactdeséchantillonsaveclapeau,lesmuqueusesetlesyeux.
9.Lestubesdesangetlematérielàusageuniqueayantserviàlamanipulationdoiventêtreéliminésdansdesconteneursadhocdestinésàl'incinération.
10.Lesréactifsetlesdéchetsdoiventêtreéliminésconformémentauxexigenceslocales.