TechnicalmethodswerAFigurePositivemyeloperoxidasereactionwith27FDAassubstrateinacaseofacutemyeloidleukaemiaM2jNoteapositiveAuerrodintwoblastsx1400QUANTITATIVERESULTSThepercentagesofpositive ID: 946979
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1114Myeloperoxidasecytochemistryusing2,7-fluorenediamineI.BENAVIDES1ANDD.CATOVSKYMRCLeukaemiaUnit,RoyalPostgraduateMedicalSchool,DucaneRoad,LondonW12OHSThemyeloperoxidasecytochemicalreactionhasbeenwellestablishedasavaluabletoolinthecharacterisationofvarioustypesofleucocytes.Thismethodhelpsinthedistinctionbetweenacutemyeloidleukaemias(AML)andacutelympho-blasticleukaemias(ALL)assuggestedintheFrench,American,andBritish(FAB)classification(Bennettetal.,1976).Themostcommonsubstratesusedarebenzidinederivatives(Graham,1918;Goodpasture,1919).Wehavebeenusingbenzidinedihydrochloride(BDC)accordingtoKaplow's(1965)technique.However,theknowncarcinogenicpotentialofthesesubstrateshasledtothewidespreadremovalofthesereagentsforclinicaluseintheUnitedStates,UnitedKingdom,andJapan.Inagakietal.(1976)reportedtheuseofthediaminederivativeoffluorene:2,7-fluorenediamine(2,7-FDA)asagoodsubstituteofbenzidineforthedemonstrationofmyeloperoxidaseinperipheralbloodandbonemarrowsamples.Wehavecomparedqualitativelyandquantitat-ivelybothreactionsinordertoassessobjectivelytheusefulnessofthenewreagent.MaterialandmethodFreshlymadebone-marrowfilmsfrom10caseswereused:AMLatpresentation(6),AMLinremission(1),chronicgranulocyticleukaemiainblastcrisis(CGL-BC)(1),polycythaemiarubravera(PRV)(1),andALL(2).Duplicatefilmsweretestedwith:(a)thereactionofKaplowasdetailedinDacieandLewis(1975),withBDC,and(b)thetechniqueofInagakietal.(1976)with2,7-FDA.Nolossofenzymeactivitywasobservedwhenthefilmswerestoredunfixedforupto10daysat4VC.ThetechniqueofInagakietal.(1976)wasusedwithslightmodificationsasfollows:(a)Fixative:10%formol-ethanolsolution.(b)Incubationmixture:2,7-FDA(Koch-LightiOnascholarshipfromtheBloodBank,CaraboboState,VenezuelaReceivedforpublication15May1978.TechnicalmethodsLaboratoriesLtd,Colnbrook,Bucks,England)wasusedasahydrogendonor.40mgof2,7-FDAweredissolvedin40mlofTris-HCIbuffer,pH8&6,toobtainasaturatedsolution.Afterstirringvigorouslyforfiveminutesatroomtemperature,themixturewasfilteredtoremovetheexcessofprecipitatedsubstrate.Justbeforeuse,twodropsof30%H202wereaddedtotheclearfiltrate.(c)Counterstain:50mlofGiemsasolutionpre-paredin1/15MS6rensen'sphosphatebuffer(pH68)inthefollowingproportions:1dropofGiemsastainpermlofbuffer.Themodificationsmadeontheoriginaltechniquewere:(1)Fixative:formol-ethanol,insteadofcoppersulphateor2-5%glutaraldehyde;(2)Counterstain:GiemsasolutionatpH6&8for15minutesinsteadofGiemsaatpH6-4for10minutesorCarazzi'shaematoxylin.ThesamefixativeandcounterstainwereusedintheBDCtechnique.CYTOCHEMICALREACTIONFilmswerefixedforoneminute,washedindistilledwater,andtransferredtoacoplinjarcontainingtheincubationmixture.Afterfiveminutes'incubationatroomtemperature,theywerewashedindistilledwaterforafewseconds,shakenofftoremovetheexcesswater,andcounterstainedintheGiemsasolutionfor15minutes.Finally,theywerewashed,driedintheair,andmountedusingaconventionalmountingmedium(Diatex,R.A.Lamb).TheincubationmixturewasstableformorethansixweekswhenstoredatroomtemperaturewithouttheadditionofH202.ResultsQUALITATIVEASPECTSWith2,7-FDA,theperoxidaseactivitywasexpressedinthepositivemyeloidcellsasyellowish-browngranulesinthethinpartso
fthefilmsandasblackordarkbrowngranulesinthethickerparts.Allneutrophilsandsomemonocytesshowedadistinctfinegranulation.Eosinophilsandbasophilsshowedlargepositivegranules.InAML,thepositiveblastcellshadoftensmallorfinegranules,andinsomecaseslargergranulesalongwithpositiveAuerrodswereclearlyseen(Figure).ThereactionproductappearedalwaysstrongerwhenBDCwasusedasasubstrate.However,themorphologicalrecognitionoftheblastswaseasierwhen2,7-FDAwasused.Thereactionwith2,7-FDAwas,ingeneral,cleanandneveroverwhelmedthecellsurfacewithprecipitatesorcrystals.Incontrast,withtheBDCmethod,formationofprecipitatesandcrystalswasoftenseen. Technicalmethods.w..erA+FigurePositivemyeloperoxidasereactionwith2,7-FDAassubstrateinacaseofacutemyeloidleukaemia(M2j.NoteapositiveAuerrodintwoblasts(x1400).QUANTITATIVERESULTSThepercentagesofpositivecellsusingbothtech-niqueswerecomparedinAML(Table):500cellswereexamined.Thepercentagesofpositiveblastcellsinallthecasesshowedlittledifferencebetweenthetwomethods;incases4and8,therewasahigherpercentageofpositivitywiththe2,7-FDAmethod.InthebonemarrowofPRV,thepercentageofpositivemyeloidcellswasalsoverysimilarwithbothtechniques.InALL,thereactionwasnegativeintheblastcells.DiscussionWehaveshownthatthecytochemicalmethodofInagakietal.(1976)isagoodalternativetomethodsinvolvingtheuseofbenzidineforthedemonstrationofmyeloperoxidaseatlightmicroscopy.Theobjectiontotheuseofbenzidineisitscarcinogenicpotential,andareliable,safesubstituteisrequired.Althoughthereactionisgenerallylessstrong,ithastheadvantagethatthecellscanbeseenmoreclearlybecausecrystalsandprecipitatesdonotformastheyareapttodowithbenzidine.InInagaki'soriginalpapernocomparativestudywiththeclassictechniqueofKaplowwasperformed.2,7-FDAmaybeaweakcarcinogenandhasproducedbreastcancerinrats(Griswoldetal.,1966),butitisnotregisteredasaknownorsuspectedTablePercentageofpositiveblastcellsinAMLandCGL-BCCaseClassification*2,7-FDA%BDC%1Ml78812M433-6353**M241414M428125Ml14126**MI90877Ml1-31-58CGL-BC(M5)104*FABclassification(Bennettetal.,1976)**AuerrodspositivewithbothtechniquescarcinogenbytheDepartmentofHealthandSocialSecurity.Withappropriatelaboratorypre-cautions,suchasavoidingcontactwiththeskinandavoidinginhalationwhenhandlingthedrysubstance,itisprobablysafe.2,7-FDAisnotconsideredanindustrialhazardandcanbeobtainedcommercially.Anon-carcinogenicsubstrate,theHanker-Yatesmixture(p-phenylenediamineHCI,1part;pyro-catechol,2parts)hasbeenrecommendedasanalternativetodiamino-benzidineforthedemon-strationofhorseradishperoxidase(Hankeretal.,1977).Inourhands,themyeloperoxidasereactionwasweakornegativewiththisreagentwhenthe1115 1116Technicalmethodsothertechniqueswereclearlypositive.Evenwhenthereactionwaspositive,itwasnotwelllocalisedinthecellcytoplasm.Thereforethisreagentisofnovalueinstudiesofbloodandbone-marrowsamples.ReferencesBennett,J.M.,Catovsky,D.,Daniel,M.T.,Flandrin,G.,Galton,D.A.G.,Gralnick,H.R.,andSultan,C.(1976).Proposalsfortheclassificationoftheacuteleukaemias.BritishJournalofHaematology,33,451-458.Dacie,J.V.,andLewis,S.M.(1975).PracticalHae-matology,5thedition,p.120.ChurchillLivingstone,Edinburgh.Goodpasture,E.W.(1919).Aperoxidasereac
tionwithsodiumnitroprussideandbenzidineinbloodsmearsandtissues.JournalofLaboratoryandClinicalMedicine,4,442-444.Graham,G.S.(1918).Benzidineasaperoxidasereagentforbloodsmearsandtissues.JournalofMedicalResearch,39,15-24.Griswold,D.P.(Jr),Casey,A.E.,Weisburger,E.K.,Weisburger,J.H.,andSchabel,F.M.(Jr).(1966).Onthecarcinogenicityofasingleintragastricdoseofhydrocarbons,nitrosamines,aromaticamines,dyes,coumarins,andmiscellaneouschemicalsinfemaleSprague-Dawleyrats.CancerResearch,26,619-625.Hanker,J.S.,Yates,P.E.,Metz,C.B.,andRustioni,A.(1977).Anewspecific,sensitiveandnon-carcinogenicreagentforthedemonstrationofhorseradishperoxi-dase(Letter).HistochemicalJournal,9,789-792.Inagaki,A.,Uno,S.,Yoneda,M.,andOhkawa,K.(1976).2,7-fluorenediamineand2,5-fluorenediamineasperoxidasereagentsforbloodsmears.JournalofLaboratoryandClinicalMedicine,88,334-338.Kaplow,L.S.(1965).Simplifiedmyeloperoxidasestainusingbenzidinedihydrochloride.Blood,26,215-219.LetterstotheEditorUseoflowionicstrengthsaltsolutionincompatibilitytestingTherehaverecentlybeenseveralpubli-cations,startingwiththatofLowandMesseter(1974),concerningtheuseoflowionicstrengthsaltsolution(LISS)inantibodyidentificationproceduresandwhencross-matching(WickerandWallas,1976;LincolnandDodd,1978).Otherauthorsarementionedinthetextbelow.InNovember1977,wepublishedinourregionalbooklet'TechnicalNotesonBloodTransfusion'detailsofourownLISSmethodsand,promptedbytherecentpublicationbyRossandDucie(1978),weshouldlikebrieflytorecordourfindings.Thestandardmethodsofcross-matchingthatwerecommendtohospitalbloodbanksinourregionarethosedescribedbyToveyandJenkins(1967)inTable1NumberandspecifiritiesofantibodiesexaminedAnti-D16Anti-K17Anti-Jka5Anti-C3Anti-k2Anti-E13Anti-Kpa2Anti-WraIAnti-c7Anti-Bga1Anti-;3Anti-Lea5Anti-Kna1Anti-Leb4Anti-Lan1Anti-S6Anti-VelIAnti-;2Anti-Fyi8Anti-Gya2Table2EffectofincubationtimesonperformanceofantibodiesbytheLISSantiglobulintestOrderofreactioncomparedwithLISSantiglobulintestincubationconventionalantiglobulintest___5min10min15min20minPercentageAbetterthan18283137Bequalto58656660Cnotasgoodas24733theACPBroadsheet57.ToevaluatetheuseofLISSinrapidcompatibilitytestsagainstthesestandardmethodswehaveexamined100antibodiesofdifferentspecificities,asshowninTable1.TheLISSsolutionusedwasthatdescribedbyMooreandMollison(1976),sterilisedandstoredat4°C.ThetechniquesusedwereLISSin-directantiglobulintestswith5-minute,10-minute,15-minute,and20-minuteincubationtimesandanindirectanti-globulintestusingphysiological(014M)salineincubatingfor60minutes.Alltestswereperformedbytheslidetech-nique.TheresultsareexpressedinTable2asthepercentageofantibodiesgivingreactionsbyLISSantiglobulintestswhichare(A)betterthan,(B)equalto,or(C)notasgoodastheconventionaltest.SevenseradidnotreactaswellbytheLISSantiglobulintest,usinga10-minuteincubationtime,asbytheconventionalantiglobulintest.Theseseracontainedanti-Kellantibodies(3),anti-Fya(2),anti-D,andanti-Leb.Inallsevencasesthereactionswereonlymarginallyweakerbythe10-minuteLISSantiglobulintest.Thebenefitsobtainedinincreasingtheincubationtimefrom5to10minutescanbeclearlyseenfromthetable.Further-more,twoweakanti-Kellseradidnotre-