/
JClGnPathol199043600603Useofbenzidineforhistologicaldemonstrationof JClGnPathol199043600603Useofbenzidineforhistologicaldemonstrationof

JClGnPathol199043600603Useofbenzidineforhistologicaldemonstrationof - PDF document

davis
davis . @davis
Follow
342 views
Uploaded On 2022-08-16

JClGnPathol199043600603Useofbenzidineforhistologicaldemonstrationof - PPT Presentation

601UseofbenzidineforhistologicaldemonstrationofhaemoglobininhumanbitemarksMethodsBlocksoftissuefromeachcasewereremovedfromtheareasofputativebruisingandchosentoincludenormalhealthytissueatoneendPieces ID: 937582

peroxidase masson pseudo saline masson peroxidase saline pseudo london newyork 1985 4thed churchilllivingstone neutralformalin 2nded strichrome andperls sreac amidoblackb

Share:

Link:

Embed:

Download Presentation from below link

Download Pdf The PPT/PDF document "JClGnPathol199043600603Useofbenzidinefor..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.


Presentation Transcript

JClGnPathol1990;43:600-603UseofbenzidineforhistologicaldemonstrationofhaemoglobininhumanbitemarksRTAllison,DKWhittakerAbstractMacroscopicevidenceofbruisingfromhumanbitemarksmaybeinconclusiveandroutinehistochemicalmethodsofshowingextravasatederythrocytescanbeunreliable.Leucopatentbluestain-ing,forthepresenceofperoxidase,AmidoblackB,atinctorialstainingmethodforhaemoglobin,Perls'sreac-tionforferriciron(haemosiderin),Masson-Fontanaformelanin,Masson'strichrome,aconnectivetissuestain,andthebenzidinereactionforhaemoglobinperoxidasewerecarriedoutinthreeforensiccasesandoneexperimentalcase.Amodifiedbenzidinemethodwasthemostreliableindicatorofhaemoglobinactivity,especiallywheredispersionintoextra-cellulartissueshadoccurred.Theresilienceoftheery-throcyteperoxidaseenzymetotem-peraturechangesandfixationsupportstheconceptofa"pseudo-peroxidase"inthosecells.Itisconcludedthatfreehaemoglobinfrombitemarks,orindeedotherformsofblunttrauma,maybestbeshownbythebenzidinereactionandthatexemp-tioncertificatesforuseofthisprohibitedsubstancemaybeworthpursuing.tissuesections,results.producingvividlypositiveCasehistoriesCASE1Thiswomanhadbeenrapedandmurdered.Theprobabletimeoffirstexaminationofthebodybyapathologistwaswithin12hoursofdeath.Rigormortiswaspresentandapostmortemexaminationwasperformedthesameday.Althoughtherewasnodetailedwrittenrecordinthemortuaryoftheconditionsunderwhichthebodywasstored,itwasreportedthatthebodywasfrozento-16°Candthawedatleasttwicebeforethetissuedescribedbelowwasexcised.Seventeendaysafterdeathoneofus(DKW)examinedthebodyforthepresenceofbitemarks-aninjurythatcouldhavebeenconsistentwithpreviousattacksbyanemergingsuspect.Bruisesandmildabrasionsweredetectedaroundthenippleoftheleftbreast.Siliconeimpressionsweremadeofthebreastbeforeandafterthawingofthebodyforreasonstobedescribedelsewhere.Oneweeklaterandfol-lowingthawingoncemore,theentirebreastwasamputatedformicroscopicexamination.Itwasimmersedin1000neutralformalin-saline.Bitemarksinhumantissuemayproducelacerationoftheepidermis,butintheabsenceofpenetrationoftheskin,arevisiblebecauseofbruisingcausedbythebitingedgeoftheteeth,'orbysuctionorpressureonthesofttissues.Abruiseistheescapeofbloodintothetissuesofalivingpersonfollowingruptureofvessels,usuallycapillaries,bytheapplicationofabluntforce.3Theellipticalshapeofahumanbitemarkandthemarkscausedbyindividualteethusuallymakerecognitionstraightforward,buttherearecaseswhenitmaybenecessaryformedicolegalreasonstoprovethatdiscolourationofthetissuesatthesiteofpressureofatooth,orteeth,isduetoextravasationofbloodandnotsomeothercausesuchaspigmentation.Extravasatederythrocyteswillusuallybedemonstrableusingstandardhistochemicaltests,butextravasationofbloodcannotalwaysbediagnosedbytheidentificationofintacterythrocytes,andPerls'sreactionforhaemosiderinmaybenegative.InarecentcaseasuspicionofbruisingfromabitecouldnotbeconfirmedhistochemicallyandasalastresortthebenzidinetechniquewasappliedtoCASE2An82yearoldwomanhadbeenattackedandmurderedathome.Examinationofthebodyshowedalargebruiseontheouterthigh.Alargeareaofthistissue,togetherwithadjacentmuscle,wasremovedandfixedin10%neutralformalin-saline.Itwassubsequentlyestablishedthatshehadbeentakingpro-phylacticanticoagulants.CASE3Thisyoungwomansustainedavicious,fatalsexualattack,andthebodyfeaturedmanybitemarks.Oneofthesewasexcisedfromtheelbow,fixedin1000neutralformalin-saline,andparaffinwaxsectionsprepared.EXPERIMENTA

LSTUDYAnareaofbruisingwasexcisedfromthethighofafemalecadaver,frozen,andstoredat-16°C,thawedtoroomtemperaturefortwohours,refrozenunderthesameconditions,andfinallythawedagainandfixedin10%neutralformalin-saline.Thiswasanattempttoreconstructthefreezinghistoryofthetis-suesexcisedincase1insofarastheywereknown.UniversityofWalesCollegeofMedicineDentalSchool,HeathPark,CardiffDepartmentofOralSurgery,Medicine,andPathologyRTAllisonDepartmentofBasicDentalScienceDKWhittakerCorrespondenceto:RTAllisonAcceptedforpublication12January1990600 601UseofbenzidineforhistologicaldemonstrationofhaemoglobininhumanbitemarksMethodsBlocksoftissuefromeachcasewereremovedfromtheareasofputativebruisingandchosentoincludenormalhealthytissueatoneend.Piecesoftissuedistantfromthemacro-scopicallybruisedareaswereselectedforcon-trolpurposes.FixedtissuewasprocessedtoparaffinwaxovernightusingtheHypercentre(ShandonScientificLtd,Runcorn,Cheshire).Theonlyunusualfeatureoftheprocessingcyclewastheinclusionofphenol-formalininthefirsttwostages,asrecommendedbySlid-dersandHopwood.'Sections(4gim)werecutandstainedbythefollowingstandardmethods5:Leucopatentblue;AmidoblackB;Perls'sreaction;Masson-Fontana;Masson'strichrome.ThebenzidinemethodofPickworth,asgivenbyPearse,6wasmodifiedbyusingthereagentinaCoplinjar,takingallnecessaryprecautionstoprotecttheoperatorandtheenvironmentfromthecarcinogen.ResultsAreascorrespondingtomacroscopicbruisingwereoftendenudedofepithelium(fig1).Furthermore,thedermisbeneaththeseareas,whetherornotitwascoveredbyepithelium,showedcondensationofsomecollagenfibres,visualisedasanincreasedeosinophilia,andoedematousspacesbetweenotherfibregroups.ThesefeatureswerenotpresentintheFigure1Areolarregionfrombreastofcase1showingareasoflossofepithelialcoveringandcondensationofconnectivetissuecompatiblewithbitemarks.Thereisnoevidenceofintactredbloodcellsorhaemosiderin.(Haematoxylinandeosin.)Figure2Sectionadjacenttothatillustratedinfig1,stainedbythemodifiedPickworth'sbenzidinetechniqueandshowingapositiveparticulatereaction. Allison,Whittakercontroltissues.Tissuethathadbeenfrozenandthawedandhadthereforereceiveddelayedfixationshowedquitereasonablemicroscopicmorphologicaldetail-inmostcasesnucleiwerestillintactandwellstained.Nointactredbloodcellswereseen,however,eitherwithinvesselsorthesurroundingcon-nectivetissuesfrombothcases1and3andfromtheexperimentalstudy,althoughtheywerepresentinabundanceincase2.TheLison-DunnLeucopatentblueVmethod,Amidoblack1OB,andPerls'sreac-tionproducedacompletelynegativereactioninthesuspectareas:controlsectionswerepositive.Masson-Fontanastainingshowedthepresenceofargentaffinpigment,presum-ablymelanin,inthebasallayersoftheepi-thelium,withnoincreaseinfall-outintheareasofsuspectedbites.TheMassontri-chrometechniqueservedtoenhancethemor-phologicalfeaturesinthesectionsstainedwithhaematoxylinandeosin.Sectionsfromcases1and3andtheexperimentaltissuesstainedbyamodifiedPickworth'sbenzidinemethodshowedafinelypigmentedbluepositivityinalltheareasfromwhichepitheliumhadbeenden-udedmicroscopicallyandwhichshowedevidenceofbruisingmacroscopically(fig2).Thisstainingwasseenonlyinthoseareasandconfirmedthattherewerenointactredbloodcellspresentinthefreezethawedtissue.Redbloodcellswerestainedinthecontroltissues.Thebenzidinemethodwasnegativeincase2.DiscussionItisrarelynecessarytoshowthepresenceofhaemoglobinotherthaninredbloodcells.Lilliesuggeststhatonoccasionfreehaemoglobinmaybefo

undinthekidneyinvarioushaemoglobinurias.7Methodsforthedemonstrationofredbloodcells,orthehaemoglobincontainedwithin,dependoneitherthelipidcontentofthecellenvelope(Baker'sacidhaematein)orthepresenceoftheperoxidaseenzyme.Haemoglobinperoxidaseisconsideredbymanyauthoritiestobearelativelystableenzyme,resistanttobothheatingandtochloroform/methanolextraction.8Thebasisofcommonmethodstoshowperoxidaseactivitydependsontheenzyme'scapacityto-catalysetheoxidationofaromaticaminesandphenolsbyhydrogenperoxide,givingrisetodyeforma-tionatthesiteofreactionbyavarietyofchemicalprocesses,suchaspolymerisation,precipitation,reduction.Benzidine,anaph-thol,andleucodyesareexamples.Lilliecategorisesthreetypesofperoxidase':thehighlylabiletissueperoxidases,therelativelystablemyeloperoxidasesofthegranulocyteseries,andtheheatandfixativestableperoxidaseofhaemoglobin.Theterm"pseudo-peroxidase"hasbeenassignedbysomeworkerstothislattervariant,probablybyBarkaandAnderson,9whostatethat,"themethodsrecommendedforthedemonstrationofhaemoglobinperoxidasearebasedonthisconceptofpseudo-peroxidaseactivity".Pearseconsidersthatmethodsforhaemoglobinperoxidasewereintendedprimarilytoshowthepresenceofredbloodcellsbyvirtueoftheirhaemoglobincontentandthatnoneoftheavailablemethodswasofsufficientlyhighspecificitytoconfirmhaemoglobinoutsideredbloodcells.6Hedididentify"thermostabletissueperoxidases"whichappearedduringautolysisandgaveapositiveresultwithben-zidine.Lisonstatedthathisleucobluemethoddidnotstainsuchmaterial.10Pearsedidnotconsiderthesetissueperoxidasestobeofanyimportanceandthatthe"pseudo-peroxidase"reactionscouldbetakenasindicativeofhaemoglobinorhaemochromogenpigment.Stevensalsomakesabaldstatementthattissueperoxidasesmaybeshown."AlthoughGomoriconsideredhiszincpatentmethodstobesuperiorforthedemonstrationofhaemoglobin,heconcededthatonlyintacthaemoglobin,andnotanyprotein-freederivatives,stainedwiththemethod.'2Ithasbeenproposedthatthecolouredreactionproductofbenzidineoxidationisatwostageevent,9withtheinitialproductionofbenzidineblue,anintensethoughunstablecompound,followedbyfullpolymerisationandtheproductionofthestablebutlessintensebenzidinebrown.Thisisanattractiveproposi-tion,butasearchofthetextbooksshowsthatalmostwithoutexceptionauthorsciteablue,black,orgreyreactionproductastheendpointofperoxidasedemonstrationusingbenzidine.Thisisalsotheauthors'experience,andeventhoughtheintermediateproductbenzidineblueisreputedtobestablefora"periodofweeks"only(sic),sectionsinourpossessionhaveremainedthatcolourforverymuchlonger.Leucopatentblueisproducedvianascenthydrogenreductionofpatentblueandsub-sequentoxidationusingendogenousperoxi-daseasthecatalyticsourceofhydrogenperoxidereduction.TinctorialmethodsarebelievedbyBancroftandStevenstoshowthepresenceofhaemoglobinasopposedtoassociatedperoxi-dase."ThesemethodsincludetheDunnThompsonmodificationofvanGieson's"method,Lendrum'shighlyselectiveKitonredtechnique,'4andtheAmidoblacklOBmethodofPutchlerandSweat.'5VanGieson'smethodsarewellknowntoberatecontrolledstructureaffinitymethods.TheAmidoblackmethodisclaimedtobehighlyselectiveforhaemoglobin(andelastin)becauseoftheahelicalstructureoftheglobinmoleculeandbecausemostothertissuesitesbecomeblockedbytissue/tannicacid/phosphmolybdicacidcomplexes.TheoriginatorsofthetechniquealsoplacegreatimportanceontheuseofZenker'sfixativetoenhanceselectivity,althoughthisisdisputedbyothers.'6Lendrum,anexpertintinctorialmeth

ods,considersthatthehighestselectivityisachievedusingthebenzidinemethodsandthatonlyintacterythrocytesandnotthosefromwhichhaemoglobinhasleachedafterdeath,showausefulaffinityfordyes.'4Inourcasesmicroscopicchangesofconden-602 603UeofbenzidineforhistologicaldemonstrationofhaemoglobininhumanbitemarkssationofcollagenfibresandthepresenceofoedemaconfirmthefindingsofMillington.Thefailureofbenzidinetostainredbloodcellsincase2ispuzzling.Thebodywasexaminedwithinafewhoursofdeath,thebruise,whichwasextremelybloody,wasexcisedandimmer-sion-fixedinformalin-salineimmediately,andthevictimhadbeentakingwarfarin.Withoutfurtherdetailedexperimentationitwouldbespeculativetosuggestwhetheranyorallofthesefactorsaccountedforthenegativestain-ingresults.Intheothercasesandtheexperimentalmaterialthelossofhaemoglobinandotherconstituentsfromtheerythrocytesafterdeathmayhavebeenenhancedbythefreezethawprocesstowhichthetissuewassubjected.OurresultsconfirmtheopinionsofLendrum'4andPearse6thattinctorialmethodsareoflittleuseinthedemonstrationofhaemoglobinanditsassociatedmetaboliteswhenfreeintissue.Theapparentresilienceofaperoxidaseenzymetoheatingandfixationsuggeststhattheparticularoneassociatedwiththeerythrocytedeservestobeconsideredseparatelyfromothertissueperoxidases.Al-thoughnotaltogetheranappropriateterm,ourfindingstendtosupporttheconceptofa"pseudo-peroxidase"inerythrocytes.Ourfindingsalsoconfirmthatbenzidinemethodsarethemostsensitiveindicatorsofhaemoglobinperoxidaseactivity,especiallywherethesesubstancesaredispersedinextra-cellulartissues.Unfortunately,fromahistochemicalview-point,benzidineisaknowncarcinogenandaprohibitedsubstanceintheUnitedKingdomandmanyothercountries.NegotiationsarecurrentlyunderwaywiththeHealthandSafetyExecutiveintheUnitedKingdomforanexemptioncertificatesothatthesubstancemaybeimportedbytheauthorsandusedforthisspecificpurpose.Currently,alicenceisrequiredfortheimportationanduseofben-zidineiftheconcentrationisgreaterthan0-10%(ChapmanI,November1989,personalcommunication).1MillingtonPF.Histologicalstudiesofskincarryingbitemarks.JForensicSciSoc1973;14:239-40.2KyddWL.Quantitativeanalysisofforcesonthetongue.JDentRes1956;35:171-4.3PolsonCJ,GeeDJ,KnightB.Injuries:GeneralFeatures.In:Theessentialsofforensicmedicine.4thed.Oxford:PergamonPress,1985:97.4SliddersW,HopwoodD.Bufferedphenolformaldehyde(pH7-0andpH55):improvedfixationinanenclosedtissueprocessor.MedLabSci1989;46:74-6.5CullingCFA,AllisonRT,BarrWT.Cellularpathologytechnique.4thed.London:Butterworths,1985.6PearseAGE.Histochemistry.Theoreticalandapplied.Volume2.4thed.London:ChurchillLivingstone,1985.7LillieRD.Histopathologictechniqueandpracticalhisto-chemistry.3rded.NewYork:McGraw-Hill,1965.8BurstoneMS.Enzymehistochemistry.NewYork:AcademicPress,1962.9BarkaT,AndersonPJ.Histochemistry.NewYork:HarperandRow,1963.10LisonL.Histochemieetcytochimieanimales.2nded.Paris:Gauthier-Villars,1953.11StevensA.Theoryandpracticeofhistologicaltechniques.2nded.London:ChurchillLivingstone,1982.12GomoriG.Microscopichistochemistry.Chicago:UniversityofChicagoPress,1952.13DunnRE,ThompsonEC.Newhaemoglobinstainforhistologicuse;slightlymodifiedvanGiesonstain.Pathol1945;39:49-50.14LendrumAC.Thestainingoferythrocytesintissuesec-tions.JPatholBacteriol1949;61:443-8.15PutchlerM,SweatF.Amidoblackasastainforhaemoglobin.ArchivesofPathology1962;73:245-9.16BancroftJD,CookHC.Manualofhistologicaltechniques.London:ChurchillLivingstone,198

Related Contents


Next Show more