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:

LukeDroney2016

• 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:

LukeDroney2016

• 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.

LukeDroney2016

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.

LukeDroney2016

1.5.3.2.1.Magneticbeadseparation

• Utilisesparamagneticbeadsconjugatedtoanantibodyofinterest(e.gCD19)

• Mixantibodyconjugatedbeadswithsample:

• Applymagneticfieldtothecolumncontainingthesample:

LukeDroney2016

• Cellsofinterestareisolatedbycollectingafterremovalofthemagneticfield

• ‘Positiveselection’–targetcellsofinterest.

• ‘Negativeselection’–targetothercells,allowcellsofinteresttopassby(e.genrichTcellsbyusinganti-CD19)

• Positive/negativeselectioncanbeappliedsequentiallytotargetcellsofinterest.

LukeDroney2016

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.

LukeDroney2016

• 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?

LukeDroney2016

• 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’?

LukeDroney2016

• 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.

LukeDroney2016

§ 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.

LukeDroney2016

• PercollconsistsofcolloidalsilicaparticlescoatedwithPVP.Thedensityisvariablethereforeallowsmorenuancedseparationofcellsandotherparticlesfromheterogenousspecimens.Percolldensitycentrifugationseparatescellsonthebasisofdensity(isopycnicseparation).

8. Outlineamethodbywhichthefollowingcellsubtypescouldbeisolated/enriched:

a. Haematopoieticstemcellsforautograft/allograft

b. BcellsforB-cellcrossmatch

c. TcellsforTcellcrossmatch