sandison ’ s rehydrating solution for histological analysis Technical note: Preparation improvement of charred cadaveric viscerausing Forensic Science International

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using sandison ’s rehydrating solution for histological analysis

Guendalina Gentile

^a

, Alessio Battistini

^a

, Salvatore Andreola

^a

, Michele Boracchi

^a

, Matteo Marchesi

^b

, Stefano Tambuzzi

^a

, Riccardo Zoja

^a,

*

aDipartimentodiScienzeBiomedicheperlaSalute,SezionediMedicinaLegaleedelleAssicurazioni,UniversitàdegliStudidiMilano,ViaLuigiMangiagalli, 37,20133Milano,Italy

bAziendaSocio-SanitariaTerritorialePapaGiovanniXXIIIPiazzaOMS,1Bergamo,Italy

ARTICLE INFO

Articlehistory:

Received19July2019

Receivedinrevisedform7October2019 Accepted14November2019

Availableonline16November2019

Keywords:

Charring

Hyperthermallesivity Sandison”srehydratingsolution Forensicpathology

Histology

ABSTRACT

Inforensicevaluationofcharredcorpses,internaldetrimentalsignsmayresultasmoresignificantof thoseobservedduringexternalexaminationandisoftenarduoustostateifavictimwasexposedtofire beforeorafterdeath.Whenthehistologicalanalysisoftheremaininginternalvisceraisnecessary,the massivedestructioncausedbythelesion,thecharringandthecoarctationofthesamplesdon'tallowto givefurtherinformationortodeterminetheremainingorganiccomponentsoftheviscera.Thislimitis determinedbytheintrinsiccharacteristicsofthisthermallesivityofself-maintenanceevenafterthe exitusofthesubject,worseningtheinitialdetrimentalframework.TheAuthors,withthepurposeof improving the microscopic visualization of the samples collected from cadavers with peculiar deterioration, asincase ofcarbonization,suggesttheuseofaspecifictechnicalprotocolbasedon theuseofSandison'srehydratingsolutionsince thesamplestreatedwiththissolutionshowed,at microscopicexamination,asubstantialhistological-morphologicalimprovement.

1.Introduction

Charringisthemostseverestageofthelesionscausedbythe expositionofthetissuestoveryhightemperaturesthroughthe directeffectofaﬂame.Generally,thisdetrimentalmeancausesa darkeningoftheskinandadryaspectofitssurface,sometimes leaving spot-size areas untouched [1]. The cadaver shows the characteristicaspectofthe“ﬁghter”duetotheforcedmuscular- tendinouscontractionandself-amputationofthelimbsispossible [2].Whencharringisextendedtothedeeptissues[3]avolumetric reductionoftheinternalorganscausedbydehydration,lesionsdue toexpansionofintracavitarygassesandcoagulativecataractcanbe observed.

Autopsy examination on these cadavers is arduous under severalaspects,suchasforpersonalidentiﬁcation,forchronologi- caldeterminationofthelesionsobserved–inordertocollocate themante-orpostmortem-,forstatingthedynamicoftheevents inordertodiscriminatebetweenaccident,suicide[4]homicide/

cadaver’sdestruction[5] and,ﬁnally,for identifyingthetypeof propellantusedasaprimer[2].

On the charredcorpse, themicroscopic examinationcan be particularly difﬁcult since not only the fragile, disrupted and extremely dry tissues can be easily lost during standard histological processing, but also because the few histological slidesobtainedarecharacterizedbyfrequenttechnicalartifacts.

TheAuthorshaveapplied,forthefirsttimeever,theSandison’s rehydratingsolutiononcharredcadaverictissuesinordertoassess whetheritsapplicationcouldimprovethehistologicalarchitecture ofbothcadavericskinandinternalorgans.Theprocedureprovedto be useful and effective, allowing us to obtain higher quality histologicalsections-reductionofthedestructivethermaleffects -comparedtotheequivalentonesobtainedfollowingthetissue fixationonlyinformalin.Inthisstudy,therefore,wepresentthe microscopicfindingsachieved after theapplicationofthistechnique.

2.Materialsandmethods

Wehaveconsidered22charredcorpses(Fig.1)thatunderwent judicialautopsyattheBureauofLegalMedicineoftheUniversityof MilanbetweenJanuary2016andJune2018.

* Correspondingauthorat:SezionediMedicinaLegale,UniversitàdegliStudi,ViaLuigiMangiagalli,37,20133Milano,Italy.

E-mailaddress:[email protected](R.Zoja).

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Thevictims,withan agerangefrom 21and 86 years,wereall Caucasianexception made for one corpseof Negroid race. The corpsesenrolledinthestudybelongedto18malesand4females deceasedbecauseofaccidentsin14cases,suicidein5casesand homicideintheremaining3cases.Onthewhole,wesampled59 fragmentsofskin(9fromthescalp;3fromtheneck;12ofthoracic skin;6ofabdominalskin;17ofskinoftheupperlimbsand12of skinofthelowerlimbs)and72fragmentsofinternalorgans.In bothcases,thesamplingwasperformedincorrespondenceofthe areasaffectedbymacroscopiclesionscaused byexcessiveheat.

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The 131 biologicalsamples,withdimensions of2.00.5cm, weresamplesindouble(withatotalamountof262samples)and underwenttwodifferentprocedures:thefirsthalf(samplesA)was directlyfixated in 10 %buffered formalin while the otherhalf (samplesB)waspreviouslytreatedforthreedayswithSandison’s rehydratingsolutionandthenfixatedin10%bufferedformalin.

After fixation, all samples were dehydrated with standard proceduresfor20hinanautomatichistologicalprocessorthanks toincreasingethanol solutions, clarifiedwithxylene substitute andembeddedinparaffinwithhighmeltigpoint(6558C).From allthesampleswecutout2-

m

^-thick^slides^by^means^of^a^Reichert

microtomeandwestainedoneofthemwithH&Estainingandthe otheronewithMasson-Goldner’strichrometechnique.Thelatter histochemicalstaining,beingabletowelldifferentiateconnective tissuefrommusculartissue,wasusedinordertobetterevaluate thepotentialrehydratingeffectofSandison’ssolution.Finally,the obtainedslideswereobservedwithaLeicaDMRmicroscopeand the most signiﬁcative areas were photographed with a Leica DC300Fdigitalcamera.

3.Results

Microscopicobservationofcharredtissuestreatedwiththetwo differentprocedures(GroupAwithstandardﬁxationandGroupB pretreated with Sandison’s rehydrating solution) has revealed importantresults.

Samples belonging to Group A showed morphological and structuralalterationstypicalofthistypeoflesivity,suchasvarious artifactsresultingfromdrying/charringof thestructures,wide- spreaddepositsofblackishmatter,structuralirregularities,wide areasofcompletedestructionofthepreparedslides,detachment andlossofseveralportionsofhistologicalslidesandalteredtissue stainability.

Viceversa,samplesbelongingtoGroupBshowedasigniﬁcant improvementof thegeneralhistologicalarchitecture,lackingof thetypicaldestructiveeffectscharacterizingthiskindoflesion.

The main morphological and structural differences between thesetwogroupsarereporteddividedinthreemajorcategories:

tegument,parenchymatousvisceraandholloworgans.

3.1.Tegument

Weanalyzedsamplesofskin

In formalin, presence of wide areas of charring of the epithelium, of the dermis, of the superﬁcial portions of the subcutaneousadipose tissue and of the tissue surrounding the muscles. In few spared areas, epithelium with marked coagulativeeffects,partialdetachmentofthedermalconnective tissueandseparationofthemuscularﬁbers.Aseverecoagulation oftheglandsoftheadnexacouldbenotedalongwithanalteration instainability(pronouncedlybasophilic)withabrownish-blackish coloration,widelaceratedareasandextensiveproteincoagulation.

Inthecontextofmusculartissue,severalcontractionbandnecrosis areascouldbeobserved.Pre-treatmentwithSandison’srehydrating solution showed a visible improvement of the general morphologyofthetissue:theepitheliumresultedtobeconserved andwellrecognizablenonethelessamarkeddissociationof the connective fibers of the dermis could be noted. Sub-epithelial connectivetissueshowedfewripplesandthevascularstructures resulted to be recognizable. Only focal areas of muscle fibers degenerative phenomena could be highlighted along with a modest volumetric reduction of the fibers themselves and confluentpyknoticnuclei(Fig.2).

Fig.1.InA:macroscopicoverviewofacadaverwithcompletecarbonizationofthebodyandlossofthelimbs.InB:aparticularofthefragmentsofinternalviscera(avictimof charringinacarduetoaccidentalexplosionofthegastank).

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3.2.Parenchymatousviscera

Weusedsamplesofbrain,cerebellum,duramater,lung,liver, spleenandkidney.

3.2.1.Brain

Informalin,cerebraltissuecouldnotbehistologicallyevaluated due to marked structural alterations caused by coagulation, charring, general fragmentation of the tissue, extensive peri- cellularvacuolizationandmarkednuclearpyknosis.Pre-treatment withSandison’srehydratingsolutionshowedaclearimprovement ofthegeneralhistologicalarchitectureofthetissue,withagood degree of conservation of the structures: vacuolization disap- pearedandthecoagulativealterationsofthewhitemattercould notbeobservedanymore.

3.2.2.Cerebellum

Informalin,modestpreservationofthecellularandhistological architecture, with several lacerations of the tissue and rough vacuolizationoftheinternalareas.Pre-treatmentwithSandison’s rehydratingsolutioneliminatedlacerationsandheatbubbles;the cellular component resulted to be recognizable nevertheless homogenizationandnuclearpyknosiswerestillobservable.The lossof fewnuclearstructuresofthenervouscellscouldstill be highlighted(Fig.3a).

3.2.3.Duramater

In formalin, marked detachment, severe alteration of the structural morphology and coagulative degeneration of the connective ﬁbers along with nuclear pyknosis of severe stage.

Pre-treatmentwithSandison’srehydratingsolutiondidn’tsigniﬁ- cantlyimprovethehisto-architectural aspectsince severealter- ationscausedbyheatcouldstillbenotedintheproteinstructures alongwithcoagulativedegenerationoftheconnectivetissuesand nuclearpyknosis.However,cellularmorphologycouldbeconsid- eredas improved ifcompared to thesamples solely ﬁxatedin formalin.

3.2.4.Lungs

Informalin,severecompletecarbonizationofthepleurallayer and ofthesuperﬁcialparenchyma,coagulationandpyknosisof the underlining pulmonary tissue, wide hemorrhagic intra- alveolar foci, restriction of the alveolar septs anddiffuseheat bubblesinthesub-pleuraltissue.Alveolarstructuresappearedto becompressedandsomealveolarwallscollapsed.Pre-treatment withSandison’srehydratingsolutionshowedanimprovementin the morphological structuresince thepleural surfacewasstill recognizableneverthelesstheeffectsofheatarestillobservable (proteincoagulationandnuclear pyknosis).A similarimprove- mentcouldbeobservedalsointheparenchymatousareawhere thealveolar structureswerestillrecognizablenevertheless the alveolar spacesresulted tobedilatated of variable dimensions duetoheat alterations.The compactaspectof thealveolithat could be noted in samples ﬁxated in formal is no longer detectable(Fig.3b).

3.2.5.Liver

Informalin,completecarbonizationoftheGlisson’scapsuleand of itsouterlayer;severecompressionand fragmentationofthe parenchyma,withrareisolatedfociofpreservedhepatocytesand of isolated areas of steatosis without alterations due to heat phenomena.Pre-treatmentwithSandison’srehydratingsolution allowedustoobserveamodestimprovementofthehistological structureandarestorationofthemorphologicalarchitectureofthe liver.Thevacuolarstructuresinthesamplescouldbeidentiﬁedas intra-cellular,anevidenceconsistentwiththediffusesteatosisof the organ. Only some isolated foci of hepatocytes were still characterizedbycytoplasmaticalterationsandnuclearpyknoses causedbyheat.

3.2.6.Spleen

Informalin,thecapsulewasstillrecognizablewithcoagulated hemorrhagicfociandroughlacerationsandvacuolizationofthe underlyingparenchymathatappearedtobeseverelycoagulated with extensive nuclear pyknosis. The parenchyma was also

11 75 M Accidental X X X X

12 56 M Suicide X X X X X X X

13 23 M Accidental X X X

14 44 M Accidental X

21 56 M Accidental X X

* includesseveralsitesoflesionswithcharredareas.

x includesothercharredsites:duramater,cerebellum,aorta,esophagus,bowelestomach.

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characterizedbythepresenceofseptsformedbyagglomeration ofalteredlymphoidcells.Pre-treatmentwithSandison’srehydrating solution did not show a marked improvement of the structuressincetheroughalterationsofthecapsuleandofthe

parenchyma thatcould be observed in the samples ﬁxated in formalinendured. However, somesplenic septssurrounded by lymphoid folliclesand small peri-trabecularlymphoid clusters couldberecognized(Fig.3c).

Fig.2.Skinsamples.Skindirectlyﬁxatedinformalinwithdestructivethermaleffects.H&EinAandE(100x);Masson-Goldner’strichromeinC(50x)andG(100x).Skinpre- treatedwithSandison’srehydratingsolutionsubsequentlyﬁxatedinformalin,withclearmorpho-structuralimprovement.H&EinBandF(100x);Masson-Goldner’s trichromeinDandH(100x).

Fig.3.aBrainandcerebellumsamples.Braindirectlyfixatedinformalinwithseverethermaleffects.H&EinA(100x);Masson-Goldner’strichromeinC(200x).Brainpre- treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withreductionofthedestructivethermaleffects.H&EinB(100x);Masson-Goldner’s trichromeinD(100x).Cerebellumdirectlyfixatedinformalinwithseverethermaleffects.H&EinE(50x);Masson-Goldner’strichromeinG(50x).Cerebellumpre-treated withSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withreductionofthedestructivethermaleffects.H&EinF(200x);Masson-Goldner’strichromeinH (100x).b.Duramaterandlungsamples.Duramaterdirectlyfixatedinformalinwithseverethermaleffects.H&EinA(50x);Masson-Goldner’strichromeinC(50x).Dura materpre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpartialreductionofthedestructivethermaleffects.H&EinB(100x);Masson- Goldner’strichromeinD(100x).Lungdirectlyfixatedinformalinwithseverethermaleffects.H&EinE(100x);Masson-Goldner’strichromeinG(100x).Lungpre-treatedwith Sandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpartialreductionofthedestructivethermaleffects.H&EinF(100x);Masson-Goldner’strichromeinH (100x).cLiverandspleensamples.Liverdirectlyfixatedinformalinwithseverethermaleffectsandrareisolatedfociofpreservedhepatocytes.H&EinA(50x);Masson- Goldner’strichromeinC(100x).Liverpre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpartialreductionofthedestructivethermal effects.H&EinB(50x);Masson-Goldner’strichromeinD(100x).Spleendirectlyfixatedinformalinwithdestructivethermaleffects.H&EinE(100x);Masson-Goldner’s trichromeinG(50x).Spleenpre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpoorreductionofthedestructivethermaleffects.H&Ein F(100x);Masson-Goldner’strichromeinH(100x).dKidneysamples.Kidneydirectlyfixatedinformalinwithseverethermaleffects.H&EinA(100x);Masson-Goldner’s trichromeinC(100x).Kidneypre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withmodestmorpho-structuralimprovement.H&EinB (100x);Masson-Goldner’strichromeinD(100x).

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3.2.7.Kidney

Informalin,wideareasofsub-capsularcharringwithdeposi- tionofblackishandhomogeneousmaterial.Theunderliningarea wascharacterizedbyanextendedproteincoagulationwithalmost completelossofthetubules(theareacoloredinred-orangeinthe microscopicsamples).Intheparenchymaunderneath,therenal corpusclewerestillrecognizablewithcytoplasmofhomogeneous aspectandmicroscopicvacuoles.Itwasalsoobservedtheabsence of the nuclei, the rough vacuolization of the parenchyma, the markedalterationsandtheabsenceoftheglomeruli,associated moreoverwiththeseverenarrowingoftheglomeruluscapillaries.

Pre-treatment with Sandison’s rehydrating solution did not demonstrate a marked improvement of the sub-capsular area since the diffuse pyknosis of the nuclei and the constrained parenchyma,associatedwiththevacuolizationoftherenaltissue in the more damaged areas could still be observed. Some glomeruli,however,resultedtobemoreevident(Fig.3d).

3.3.Holloworgans

We analyzed samples of esophagus, aorta, heart and small intestine.

3.3.1.Esophagus

Informalin,alterationsthatcouldbeascribedtothehardening causedbyheat,withwide fragmentationand onlysmallfociof intactmuscularwall.Pre-treatmentwithSandison’srehydrating solutionallowedtorecognizethecellularstructureseventhougha detachment of the epithelium from the basal membrane, a coarctationand fragmentation of the sub-epithelial connective tissueandnuclearpyknosisintheareassubjectedtodetachment.

In the areas close to carbonization, the epithelial structures appearedtobeconserved.

3.3.2.Aorta

Informalin,roughvacuolizationandiper-eosinophilicparallel stripes.Thistypicalartifactknownas“rollershatter”iscausedbythe excessivehardeningofthetissuethatproducesa“jump”oftheblade ofacertainamountofmicronsduringslide-cutting.Pre-treatment

with Sandison’s rehydrating solution allowed to recover the structure of theaorta and theartifact was notvisible anymore (Fig.4a).

3.3.3.Heart

Informalin,completelossofpericardialadiposetissuecaused by carbonization, marked charring, nuclear pyknosis of the endocardialmusculartissue andabsenceofthecellularoutline.

Rough vacuolization associated with coagulative alterations caused by heat were confirmed by the intense eosinophilic stainingofthecytoplasm.Widespreadfociofcontractionbundle necrosis with confluent nuclear pyknosis forming a structure similar to a chain. Pre-treatment with Sandison’s rehydrating solution did not eliminate degenerative alterations of the pericardial adipose tissue that resulted to by charred; sub- endocardial fibers were fragmented and bundle necrosis was particularlyevident.Smallfociofcellulardamagesimilartothat observedinformalinfixationcouldbedemonstrated(Fig.4b)

3.3.4.Smallintestine

In formalin,severe alterationsdue toheatthat involvedthe tissue until the muscular wall and disappearance of the sub- mucousadiposetissue.Theintestinalwallwasaffectedbyrough structuralalterationswithcharredareas,fragmentedﬁbersanda few fociofsparedwall hardlyrecognizable. Pre-treatmentwith Sandison’s rehydratingsolutionallowed topreserve thegeneral architecture of the intestinal villi nevertheless some marked coagulative alterations of the cells with pyknotic nuclei and charredmaterialcouldbestillnoted.Theentiremuscularwallwas substitutedbycharredmaterial(Fig.4c).

4.Discussion

Theuseofdifferentrehydratingsolutionsisincreasinglytaking placeinvariousforensicprocedures:foridentificationprotocolson mummified phalanges [6], in the entomologic field for the proceduresinvolvingDipteralarvae[7],oncadaversundergoing colorativo/emphysematous phenomena inorder toevaluatethe cutaneousfurrowcausebyhangingmechanicalasphyxia[8]and Fig.3.(Continued)

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oncutaneoussamplesundergoingpeculiarputrefactivephenom- ena-coriﬁcationandmummiﬁcation[9]inordertoimprovethe histologicalexamination

WehaveappliedtheSandison’s rehydratingsolution,forthe firsttimeever,tocutaneousandvisceralsamplescollectedfrom22 charredcorpses.ThehistologicalslidesobservedafterbothH&E and Masson-Goldner’s trichrome staining,showed qualitatively superiormorpho-structuralaspectscomparedtothose obtained by routine fixation with formalin. Actually, the Sandison’s rehydrating solutionby means of an emulsifier in it - sodium carbonateNa2CO3–penetratesdeepandhomogeneouslyintothe cadaverictissues,bymeansofasolvent-96%ethanol-stabilizes thetissuesandpreventstheircollapseand,finally,bymeansofa fixative-formalin-inactivatesthebacteria[9].Forthesereasons, thistechniquehasdetermineda goodimprovementbothofthe tissuemorphologyandofitsstainability,withtherestorationof thestructures andpartial eliminationof theartifactsrelated to hyper-thermallesivity,well-knowndestructivelesivity.

On the whole, compared to the samples obtained byroutine ﬁxationwithformalin,itsuseonskinsampleshasproducedaclear improvementofthetissuemorphology,onparenchymatousviscerait haspartially eliminatedthealterationscaused byseverecharringwith adiscreetimprovementandonholloworgansitallowedamoderate regeneration of histological structures. In detail, the Sandison’s rehydratingsolutionprovedtobeparticularlyeffectiveonskin,brain andaortasamples;itallowedtoobtainapartialhistomorphological restorationofduramater,esophagus,lung,heartliverandkidney samples;itdidnotallowtoobtainsigniﬁcantimprovementsofspleen and small intestine samples, in which signs of heat protein coagulation,coarctationsandfociofnuclearpyknosispersisted.

Consideringthemicroscopicﬁndingsachievedinthetwogroups, the samplesobtained afterpre-treatmentwithSandison’srehydrating solutionareofhigherqualitythanthoseﬁxeddirectlyinformalin.

Therefore,weconsidertheuseofthisrehydratingsolutiononcharred cadaveric tissuesto be valid, neverthelessit wasobserved a less morpho-structuralimprovementonfewviscera.Actually,inallcases, Fig.4. aEsophagusandaortasamples.Esophagusdirectlyfixatedinformalinwithseverethermaleffects.H&EinA(50x);Masson-Goldner’strichromeinC(50x).Esophagus pre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpartialreductionofthedestructivethermaleffects.H&EinB(100x);Masson- Goldner’strichromeinD(100x).Aortadirectlyfixatedinformalinwithseverethermaleffects.H&EinE(100x);Masson-Goldner’strichromeinG(100x).Aortapre-treated withSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withmodestmorpho-structuralimprovement.H&EinF(50x);Masson-Goldner’strichromeinH(50x).

bHeartsamples.Heartdirectlyfixatedinformalinwithdestructivethermaleffects.H&EinAandE(100x);Masson-Goldner’strichromeinC(200x)andG(50x).Skinpre- treatedwithSandison’srehydratingsolutionsubsequentlyfixatedinformalin,withpartialreductionofthedestructivethermaleffectsandpersistenceofcontractionband necrosisareas.H&EinB(50x)andF(100x);Masson-Goldner’strichromeinD(200x)andH(400x).cSmallintestinesamples.Smallintestinedirectlyfixatedinformalinwith destructivethermaleffects.H&EinA(100x);Masson-Goldner’strichromeinC(50x).Smallintestinepre-treatedwithSandison’srehydratingsolutionsubsequentlyfixatedin formalin,withpoorreductionofthedestructivethermaleffects.H&EinB(100x);Masson-Goldner’strichromeinD(100x).

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responsibleforthecontentandwritingofthispaper.

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