【astm標準】e 2524 - 08 (2013)

1、Designation:E2524?08(Reapproved2013)StardTestMethodfAnalysisofHemolyticPropertiesofNanoparticles1ThisstardisissuedunderthefixeddesignationE2524thenumberimmediatelyfollowingthedesignationindicatestheyearofiginaladoptionin

2、thecaseofrevisiontheyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperepsilon()indicatesaneditialchangesincethelastrevisionreapproval.1.Scope1.1Thistestmethodcoversassessingtheeffectofnanopart

3、iculatematerialsontheintegrityofredbloodcells.1.2Thistestmethodusesdilutedwholebloodincubatedwithnanoparticulatematerialthehemoglobinreleasedfromdamagedredbloodcellsisdetermined.1.3ThistestmethodissimilartoPracticeF756wi

4、ththevolumesreducedtoaccommodatenanoparticulatematerial.1.4Thistestmethodispartoftheinvitropreclinicalacterizationisimptantfnanoparticulatematerialthatwillcontactthebloodinmedicalapplications.1.5ThevaluesstatedinSIunitsa

5、retoberegardedasstard.Nootherunitsofmeasurementareincludedinthisstard.1.6Thisstarddoesnotpurpttoaddressallofthesafetyconcernsifanyassociatedwithitsuse.Itistheresponsibilityoftheuserofthisstardtoestablishappropriatesafety

6、healthpracticesdeterminetheapplicabilityofregulatylimitationspritouse.2.ReferencedDocuments2.1ASTMStards:2F748PracticefingGenericBiologicalTestMethodsfMaterialsDevicesF756PracticefAssessmentofHemolyticPropertiesofMateria

7、lsF1877PracticefacterizationofParticlesF1903PracticefTestingFBiologicalResponsestoParticlesInVitro2.2ISOStard:3ISO109934BiologicalEvaluationofMedicalDevicesPart4:ionofTestsfInteractionswithBlood3.Terminology3.1Acronyms:3

8、.1.1Cal—calibrationstard3.1.2CMH—cyanmethemoglobin3.1.3DPBS—Dulbecco’sphosphatebufferedsaline3.1.4PEG—polyethyleneglycol3.1.5PFH—plasmafreehemoglobin3.1.6QC—qualitycontrols3.1.7TBH—totalbloodhemoglobin3.1.8TBHd—bloodsamp

9、ledilutedto10mg61mgmL4.SummaryofTestMethod4.1Thistestmethoddescribesaprotocolfassessingacuteinvitrodamagetoredbloodcells(thatishemolysis)causedbyexposuretonanoparticles.4.2Thistestmethodisbasedonthequantitativedeterminat

10、ionofhemoglobinreleasedintoPFHasapercentageoftheTBHconcentrationwhenbloodisexposedtonanoparticulatematerials.4.3Usinganestablishedcolimetricassay4hemoglobinitsderivativessuchassulfhemoglobinareoxidizedtomethemoglobinbyfe

11、rricyanideinthepresenceofalkali.AstableCMHconcentrationismeasuredusingaplatereaderspectrophotometersetat540nm.4.4Hemoglobinstardsareusedtocreateastardcurvecoveringtherangefrom0.025to0.8mgmLpreparequalitycontrolsamplesatl

12、ow(0.0625mgmL)(0.125mgmL)high(0.625mgmL)concentrationstomonitassayperfmance.Therequiredsamplevolumeis100Lpertestreplicate.4.5Theresultsareexpressedaspercenthemolysistoevaluatetheacuteinvitrohemolyticpropertiesofnanoparti

13、cles.5.SignificanceUse5.1ThistestmethodisoneofaseriesoftestslistedinPracticeF748ISO109934toassessthebiocompatibilityofmaterialscontactingbloodinmedicalapplications.1ThistestmethodisunderthejurisdictionofASTMCommitteeE56o

14、nNanotechnologyisthedirectresponsibilityofSubcommitteeE56.03onEnvironmentHealthSafety.CurrenteditionapprovedSept.12013.PublishedSeptember2013.iginallyapprovedin2008.Lastpreviouseditionapprovedin2008asE2524–08.DOI:10.1520

15、E252408R13.2FreferencedASTMstardsvisittheASTMwebsitewww.astm.gcontactASTMCustomerServiceat.FAnnualBookofASTMStardsvolumeinfmationrefertothestard’sDocumentSummarypageontheASTMwebsite.3AvailablefromAmericanNationalStardsIn

16、stitute(ANSI)25W.43rdSt.4thFloNewYkNY10036:www.ansi.g.4InternationalCommitteefStardizationinHaemotologyJournalofClinicalPathologyVol311978pp.139–143.Copyright?ASTMInternational100BarrHarbDrivePOBoxC700WestConshohockenPA1

17、94282959.UnitedStates1don.Ifmicroaggregatesofbloodareobservedfilterthebloodthrougha40mbloodfilter.8.6.2Takea2to3mLaliquotofthepooledbloodcentrifuge15minat800g.8.6.3Collectthesupernatant.Keepatroomtemperaturewhilepreparin

18、gthestardcurvequalitycontrolstotalhemoglobinsample.Thecollectedsupernatant(plasma)isusedtodeterminePFH.9.DeterminationofPFHTBHinNativeBloodSample9.1Add200.0LofeachcalibrationstardqualitycontrolblankCMHreagentperwellon96w

19、ellplate.UsetwowellsfeachcalibratfourwellsfeachQCtheblanksothattestsamplesarebracketedbyQCsfexampleasequencesuchas(blankCal1Cal6QC1QC2QC3testsamplesblankQC1QC2QC3).9.2PreparetheTBHsamplebycombining20.0Lofthepooledwholebl

20、ood5.0mLofCMHreagent.After15minadd200.0Ltoeachofsixwells.9.3Add100.0Lofplasma(8.6.3)tosixwellsadd100.0LofCMHreagenttoeachofthesewells.DonotaddCMHreagenttowellscontainingcalibrationstardsqualitycontrols.9.4Covertheplatewi

21、ththeplatesealergentlyshakeonaplateshaker(mediumspeedsettings2to3).9.5Readtheabsbanceat540nmtodeterminethehemoglobinconcentration.Remembertouseadilutionfactof2fthePFHsampleadilutionfactof251fTBH.IfthecalculatedPFHconcent

22、rationisbelow1mgmLproceedtothenextstep.Ifitisabove1mgmLthebloodsampleisnotsuitableftheprocedure.10.ProcedurewiththeTestMaterial10.1DilutepooledwholebloodwithCa2Mg2freeDPBStoadjustTBHconcentrationto1061mgmLbasedonresultsf

23、rom9.5.10.2Add100.0Lofsamplereagentblank(PBSotherbufferusedtodissolvenanoparticles)positivecontrolsolutionnegativecontrolsolutiontomicrocentrifugetubes.Preparesixtubesfeachunknownsamplethreetubesftheblanktwotubesftheposi

24、tivecontroltwotubesfthenegativecontrol.10.3Add700.0LofCa2Mg2freeDPBStoeachtube.10.4Add100Lofthewholebloodpreparedin10.1toeachtubeexceptfthreetubesofeachtestsample.Inthesetubesadd100LofCa2Mg2freeDPBS.Thesesamplesrepresent

25、a“minusblood”controlareusedtoevaluatepotentialinterferenceofnanomaterialwiththeassay(fexampleabsbanceatcloseto540nmreactivitywithCMHreagentsofth).10.5Covertubesgentlyrotatetomix.Ateach30minintervalobservewhetherthesample

26、ofnanoparticlesflocculatedispersesinkfloatduringtesting.NOTE3—Vtexingmaydamageerythrocytesshallbeavoided.10.6Placethetubesinawaterbathsetat3761Cincubatef3h615minmixingthesamplebyrotationevery30min.Alternativelytubesmaybe

27、incubatedonatuberotatinanincubatsetat3761C.10.7Removethetubesfromwaterbathincubat.Ifawaterbathwasuseddrythetubeswithabsbentpaper.10.8Centrifugethetubesf15minat800g.10.9Whencentrifugationiscompleteexaminethetubesrecdanyun

28、usualappearanceofthesupernatantpelletthatcanindicateadditionaldamagetotheredbloodcellsthehemoglobinadsptionofhemoglobintotheparticles.SeeFig.1.10.10InFig.1commerciallyavailablepreparationsofpolystyrenenanoparticleswithsi

29、ze20nm(Tube1)50nm(Tube2)demonstratedhemolyticactivitythatcanbeobservedbythecolofthesupernatant.Polystyrenenanoparticleswithsize80nmwerealsohemolytichowevertheyadsbedFIG.1ExampleDemonstratingtheImptanceofRecdingSampleAppe