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LukeDroney2016
PreparationofSamples
1.5.3.1.Explaintheprocedureandbeabletoperformseparationofsamplesforlaboratoryanalysis
1.5.3.1.1.Serumseparation
1.5.3.1.2.Concentrationofbodyfluids(urine,CSF)
1.5.3.1.3.Ficoll-hypaqueseparationofperipheralbloodlymphocytes
1.5.3.1.4.Preparationofacellsuspensionfromsolidtissue
1.5.3.1.5.Separationofneutrophilsfromwholeblood
1.5.3.2.Befamiliarwithadvancedcellpurificationtechniques
1.5.3.2.1.Magneticbeadseparation
1.5.3.2.2.Flowcytometry-basedsorting
1.5.3.2.3.Percollgradientdensitycentrifugation
1.5.3.1.1.Serumseparation
Serumseparation:
• Serum=nocellsorclottingfactors
• Fractionofbloodcollectedafterwholebloodallowedtoclot
• Clotremovedbycentrifugation.
• Gelcanhelpseparation
Stepstocollectingserum:
1. Collectwholebloodinserumtube
2. Allowthesampletoclotatroomtemperaturefor15-60minutes(mayvarydependingonmanufacturer).Ifleftforlongerthan60minutesmayhaemolyse.
3. Removeclotbycentrifugation(refrigerated)at1000-2000rpmfor10minutes.
4. Transfersupernatanttocleanpolypropylenetube.Aliquot,keepsampleat2-8degrees.Transport/storeat-20.Minimisefreeze/thawcycles.
Plasmaseparation:
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• Plasma=wholebloodcollectedwithanticoagulant(EDTA,citrate,heparin)i.econtainsclottingfactorsandsomecellularmaterial.
Stepstocollectingplasma:
1. CollectwholebloodinEDTA/citrate/heparinisedtube.
2. Removecellsbycentrifugationandaspiratesupernatant.
1.5.3.1.2.Concentrationofbodyfluids(urine,CSF)
Urine:
• Proteinconcentrationinurineislowerthanserum
• Thereforeconcentrationofthespecimenisrequiredforadequatesensitivity
• Methodsofconcentration:
o Ultrafiltration
o Increasedapplicationtime
o Increasedapplications
o Centrifugalultrafiltration
• Concentrationdeterminedbytotalproteincontentofthesample:
• Underconcentration–decreasedsensitivity
• Overconcentration–overloadedgelsthatmayobscuresmallbands.
CSF:
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• Similartourine,muchsmalleramountsofproteininCSF(1/100–1/200ofserum)
• Diluteserum60-foldforisoelectricfocussing
1.5.3.1.3.Ficoll-paqueseparationofperipheralbloodlymphocytes
Ficoll-Paque;
• Neutral,highlybranched,highmasshydrophilicpolysaccharide.
• Density1.077g/ml–optimisedforisolationofhumanmononuclearcells.
• 60+/-20%recoveryoflymphocytes
• SelectivelossofTandNKcellsmayoccur
• MayupregulateCD54,CD62L,CD11b
1.5.3.1.4.Preparationofacellsuspensionfromsolidtissue
1. Attentionshouldbepaidtopre-analyticalfactorssuchasflowcytometersetup/calibrationandtheuseoflocallyvalidatedpanels/monoclonalantibodies.
2. Ifcollectedlocally,thespecimenshouldbeforwardedtotheflowlaboratoryforprocessingassoonaspossible,ideallywithinonehour.IfcollectedoffsitethespecimenshouldbeprocessedandtransportedinRPMIat4degrees.
3. Attheflowlaboratory,thespecimenshouldbeminced/passedthroughsievetocreateacellsuspension.Somecentresmayuseautomatedtechniquesorenzymaticdigestion.However,thesetechniquesmayaffectcellularintegrity.
4. Cellsshouldbecounted.
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5. Insomeinstances,aviabilityassessmentcanbecarriedoutbystainingthesamplewithpropidiumiodideor7-AAD.
6. Stainthecellsuspensionwithmonoclonalantibodies(usuallyforalymphnodethiswouldbeatwo-tube‘chronicpanel’)andincubate.
7. Redcelllysis(ifsignificantbloodcontamination)–redcelllysiscanbedonepriortostaining(howit’sdoneinourlab)
8. Washtoremovelysedredcells.9. (permeabiliseandfix,thenstainwithintracellularmonoclonalsifdoingintracytoplasmic
staining).10. Fixwithpara-formaldehydesolutionifanydelayinrunningthroughflowcytometer.11. Runthesamplethroughtheflowcytometer.12. Collectandanalysedata.
Separationofcells–generalpoints:• Twomaingroups;• Basedonphysicalcriteriai.esize,shapeanddensity–filtrationandcentrifugation
techniques.• Affinitymethods–basedonbiochemicalcellsurfacecharacteristics–i.ecaptureonsolid
surfaces(beads/matrix),FACS,magnetic
Nyloncolumns–monocytesbind,usedtopurifyTcells.Immunoabsorptionassays/columnElispot
1.5.3.1.5.Separationofneutrophilsfromwholeblood
• ThedensitygradientseparationmethodisusedtoisolatehumanneutrophilsfromwholebloodusingamixtureofsodiummetrizoateandDextran500.
• Aftercollectionfromadonor,wholebloodmaybeanticoagulatedwithEDTA,citrate,orheparin.
• Sincetheyareshort-lived,neutrophilsshouldbeusedwithin2-4hoursofcollection.• Theprocedureconsistsoflayeringwholebloodoverthedensitygradientmedium,• Centrifugeat500RCFfor35minat20-25°C.Thebloodshouldseparateoutinto6distinct
bands(seebelow):plasma,monocytes,isolationmedia,neutrophils,moreisolationmedia,andtheredbloodcellpellet.Ifthesebandsarenotclear,theseparationprocesswasnotcleanandwillneedtoberepeated.
• Carefullyremovethetopthreelayers(plasma,monocytes,andisolationmedia)usingapipette.Disposeoftheselayers.
• Carefullypipettethelayerofneutrophilsandalloftheisolationmediabeneaththeneutrophils.Placethesolutionintoacleancentrifugetube.
• Resuspendneutrophils,centrifugeandlyseresidualRBCs.• Cellsarethenwashed,counted,andresuspendedtodesiredconcentration.
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1.5.3.2.1.Magneticbeadseparation
• Utilisesparamagneticbeadsconjugatedtoanantibodyofinterest(e.gCD19)
• Mixantibodyconjugatedbeadswithsample:
• Applymagneticfieldtothecolumncontainingthesample:
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• Cellsofinterestareisolatedbycollectingafterremovalofthemagneticfield
• ‘Positiveselection’–targetcellsofinterest.
• ‘Negativeselection’–targetothercells,allowcellsofinteresttopassby(e.genrichTcellsbyusinganti-CD19)
• Positive/negativeselectioncanbeappliedsequentiallytotargetcellsofinterest.
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1.5.3.2.2.Flowcytometry-basedsorting
• Individualcellsaresubjectedtolaseraspernormalflowcytometry• Afteremergingfromthenozzlethestreamisbrokenintodropletsbyvibrationofthenozzle.• Thedropletsizeissuchthateachcontainsonecell.• Achargeisassignedtoeachdropletbasedonthefluorescencequalities.• Thedropletentersanelectricfieldandisdeflecteddependingonthecharge/fluorescent
qualitiesandintoareceptacle.• Forexampleintheabovediagram:
o AsingleFITC-stainedcellwouldbeassignedapositivechargeanddeflectedtotheright,singlePE-stainedcellwouldbeassignedanegativechargeanddeflectedtotheleft.
• Fluorescence-activatedcellsortinghassomemajorlimitations,suchaslossesintheyield,therequirementforspecialtechnicalexpertise,andacomparativelylowthroughput,typicallyintherangebetween104and105cellspersecond.
1.5.3.2.3.Percollgradientdensitycentrifugation• Percollconsistsofcolloidalsilicaparticlesof15–30nmdiametercoatedwith
polyvinylpyrrolidone(PVP).Duetoitsheterogeneityinparticlesize,sedimentationoccursatdifferentrates,creatingisometricgradients.
• Percollisisotonicthroughoutthegradient,non-toxicandeasilyremovedaftercellpurification.
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• CalibrationofPercollgradientscanbesimplifiedwithdensitymarkerbeads(pre-determineddensitiesandcolours.
• Percolldensitycentrifugationutilisesisopycnicseparation,orseparationonthebasisofdensity(irrespectiveofsize).
• Eachparticlewillsedimenttoanequilibriumpositioninthegradientwherethegradientdensityisequaltothedensityoftheparticle(isopycnicposition)
• Goodformonocytes
• Rate-zonalcentrifugationseparatesparticlesonthebasisofsizeanddensityandistimedependent–Percollcanbeusedforrate-zonalcentrifugation.
Questions:
1. Aninternphonesyoutoaddananti-GADELISA.Howeveryoudiscoverthatthereisonlyplasmaavailable.Theinternisveryupsetwhentheydiscoverthatyouareunabletoperformthetestontheavailablespecimen.Explainwhy.
a. What’sthedifferencebetweenserumandplasma?
b. Howwouldyouprepareeachofthesespecimens?
• Plasmacontainsclottingfactorsandcellularelements.Theanti-GADELISAisnotvalidatedforthisspecimentype.ItisaTGA/kitrequirementthattestsonlybeperformedwiththespecimentypeforwhichthekitisvalidated.PlasmacontainselementsthatmayinterferewiththeELISA,renderingthetestredundant.
2. Whatarethedifficultiesindetectingmonoclonalproteinsbyurineproteinelectrophoresis?
a. Whymightaurineelectrophoresisbefalselynegative?
b. Howwouldyouprepareaurinesampleforelectrophoresis?
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• Urinecontainssignificantlylessproteinthanserumundernormalcircumstances.Toensureadequatesensitivityurinesamplesshouldbeconcentrated.Totalproteininthespecimenshouldbemeasuredbyconventionalmethodsthentheurineshouldbeconcentratedaccordingtotheabovetable.Iftheurineistooconcentratedthenthiscanmakethegeldifficulttointerpret.Themostcommonmethodofproteinconcentrationisultrafiltrationusinganosmoticgradient.
3. Ananatomicalpathologistfromdownstairsphonesyouaboutaflowcytometryresult.TheyhavefounddiffuselargeBcelllymphomaaffectingalymphnode.ThesamplewascollectedinRockhamptononThursdayafternoonandarrivedinBrisbaneonFriday.FlowcytometrywasperformedonSaturdaymorning.Theflowcytometry(seebelow)wasreportedasnon-diagnostic.
a. Whymighttheresultsbediscordant?
b. Howwouldyouexplainthediscrepancyinresultstothehistopathologist?
c. Whatstepsmightyoubeabletoputinplacetoincreasethediagnosticyieldforspecimenscomingfromperipheralsites?
d. Describeindetailthestepsrequiredinpreparingaresectedlymphnodeforevaluationbyflowcytometry.
e. Whyareanatomicalpathologistsalways‘downstairs’?
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• Pre-analyticalfactors:
o Needtoensureasmuchaspossiblethatthecorrectsamplehasbeenanalysed.Checkwithreferringlababoutotherspecimensthatweresentonthisday.Checkwithflowlababoutotherspecimensthatwereanalysedthatday.
o Flowshouldbeperformedassoonaspossibleafterspecimencollection.Within24hoursifpossible,definitelywithin48hours.Beyondthisperiod,viabilityislikelytobereducedandsomeorallofthecellsofinterestmaybedeadandstainnon-specifically.Thisisparticularlyimportantfortumourswithhighcellturnover.Thesampleinquestionislikelytobediscordantduetothetimetakentotransportthesampletotheflowlab.
o Decreasedviabilityissuggestedbythedegreeofnon-specificstainingandlackofforwardscatter.Aviabilityassessmentatthetimeofflowwouldhavemadethisclearer.
o Thepresenceofclotsorhaemolysismaybeindicativeofcellloss.
o Extremesoftemperatureshouldbeavoidedintransport.
o ThespecimenshouldbeprocessedASAP–within1hour.Ifthespecimenisabletobedeliveredimmediatelytothelabthentransportmoistinculturemedium.
o Ifcollectedataperipherallabthetissueshouldsubmittedtotheflowlabinthinslices(i.e2mm)andtransportedinsteriletissueculturemediumornutrientmedium(e.gRPMI)at4degrees.
o Makesurethatthesamplehasn’tbeensenttotheflowlabinformalin.
o Slicessentforflowshouldbeimmediatelyadjacenttothosesentforhistopathology.Theresultsmightbediscordantbecauseallofthegoodmaterialwassenttohistoandflowreceivedaperipheralpieceofthetissuethatdidn’tcontainadequatelymphnodecontents.
o Stepstoimprovediagnosticyieldforspecimenscomingfromperipheralsites:
§ Timelytransferofspecimenstotheflowlab.
§ Timelyprocessingofspecimensatreferringlab.
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§ Transportspecimensat4degreesinRPMI.
o SpecimenshouldberunASAPwhenitarrivesattheflowlab.
• Analyticalfactors:
o Ensurecorrectinstrumentset-upandmaintenance,QCetc.
o Prepareandstaincellsasabove.
o Ensurethattheanalysisofacquireddataisperformedcorrectlyi.ehavethecorrectpopulationsbeengated,havelargelymphocytesbeenlookedforoutsideofthelymphocytegate,havemonoclonalsdefinitelybeenadded.
• Post-analytical–checkthatthecorrectreporthasbeenenteredforthecorrectpatient.
4. Discussmethodsforcellseparationingeneralterms.
• Basedonphysicalcriteriai.esize,shapeanddensity–filtrationandcentrifugationtechniques.
• Affinitymethods–basedonbiochemicalcellsurfacecharacteristics–i.ecaptureonsolidsurfaces(beads/matrix),FACS,magnetic
5. WhatdoyouuseFicollforintheimmunologylab?
a. WhatarethelimitationsofFicoll-Paquedensitycentrifugationformalignantimmunophenotyping?
• Isolationofmononuclearcells.Lymphocytefunctionalassays.
• Ficollseparationofmononuclearcellsinvariablyleadstocellloss(particularlyTandNKcells)thereforeitisnotsuitableformalignantimmunophenotypingapplications.
• UseofFicollmayresultsinchangestoimmunophenotype.
6. Whatisthemainapplicationfordextransedimentationforintheimmunologylab?
• Isolationofneutrophilse.gforNBT,neutrophilchemotaxis,neutrophilphagocytosis
7. WhatisthedifferencebetweenFicoll-PaquedensitycentrifugationandPercolldensitycentrifugation?
• Ficollissingledensitythatisdesignedtoseparatemononuclearcellsfromgranulocytesanderythrocytes.
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• PercollconsistsofcolloidalsilicaparticlescoatedwithPVP.Thedensityisvariablethereforeallowsmorenuancedseparationofcellsandotherparticlesfromheterogenousspecimens.Percolldensitycentrifugationseparatescellsonthebasisofdensity(isopycnicseparation).
8. Outlineamethodbywhichthefollowingcellsubtypescouldbeisolated/enriched:
a. Haematopoieticstemcellsforautograft/allograft
b. BcellsforB-cellcrossmatch
c. TcellsforTcellcrossmatch