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DermNetNZ
FactsabouttheskinfromDermNetNewZealandTrust.

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Specialstainsandtests
Learningobjectives
Introduction
Specialstains
Specialtests
Activity

Learningobjectives
Listtheindicationsforspecialstainsandtests
Describethesestainsandtests

Introduction
Althoughthemajorityofskinbiopsiesaresuccessfullyprocessedusingroutineformalinfixationandstainedwithhaematoxylin&eosin
(H&E),occasionallyspecialstainsandprocessingtechniquesarerequiredfordermatopathologicaldiagnosisandclinicalmanagement.

Specialstains
RoutineH&Estainingdoesnotidentifyalltissuesubstancessincertaininflammatoryandneoplasticskindiseasesitmaybenecessary
tstainsectionswithavarietyofdifferentagentsorperformdifferentprocedures.

Stainsusedinroutinedermatopathology

H&Eofnormalskin

PASstainingoffungiinstratumcorneum Mastcells(toluidineblue)

Varioushistochemicalstainsusedtbeusedtidentifycelltypesorspecificsubstancesinsections.Thesecouldbecomplexandfickle,and
manyhavenowbeenrenderedlargelyobsoleteinroutinepracticebythedevelopmentofimmunohistochemisty.Somehistochemical
stainsarestillveryuseful.
Tissuematerialtbedemonstrated
Mucin(mucopolysaccharides)

Stainsused
PAS(periodicacidSchiff)forneutralmucin
AlcianBlueforacidmucin
Mucicarmine

Melanin

Iron(haemosiderin)

Calcium

FontanaMasson

Perl'sPrussianBlue

VonKossa
Alizarinred

Fibrin

Elasticfibres

MSB(MartiusScarletBlue)

EVG(ElasticVanGeisen)forreticulardermis
Orceinforpapillarydermis

Fat

OilRedO(Fatisdissolvedintissueprocessing,frozensectionrequired)

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Mastcells

ToluidineBlue
Giemsa

Bacteria

Gram(gramnegativeverydifficulttdemonstrate)
ZiehlNeilsonformostmycobacteria(ZNforAFB)
WadeFiteforM.Leprae

Fungi

PAS
Grocott/Gomorimethenaminesilver(GMS)

Specialtests
FrozenSections
Attimesduringasurgicalprocedureitisnecessarytaskthepathologistforarapiddiagnosistplantreatment.Thisisalmostalwaystallow
assessmentoftheresectionmarginstcheckcompletenessofexcisionincaseswheretheextentofthelesionisnoteasytseeclinicallyand
whenmajorreconstructionwillberequiredatthetimeofsurgery.Sometimesatcriticalanatomicalsites,aspecialtechniqueoffrozen
sectioncontrol(Moh'ssurgery)maybeused.
Exceptinveryrarecases,itisnotgenerallyconsideredgoodpracticetmakeaprimarydiagnosisofmalignancyonafrozensection.Thisis
particularlytrueinthecaseofpigmentedlesions,wherethediagnosis,evenonideallyprocessedparaffinsections,maybeextremely
difficult.
Frozensectionsshouldbediscussedwiththepathologistpriortcommencingsurgerytensurethatboththepathologistandatechnologist
willbeavailableattherequiredtime.
Thetissueissnapfrozent20t70Cinacoldliquidsuchasliquidnitrogen.Thefrozentissueissolidenoughtbesectionedwitha
microtomeinacryostatandthesectionsarethenpickeduponaglassslidereadyforstaining.
Thesectionsarethickerthanstandardparaffinsectionsandthetissuequalityisnotashigh.Forthisreason,tissuetakenforfrozen
sectionisalwayssubsequentlyprocessedforparaffinsectionstcheckthereport,andoccasionallyfeaturesarefoundinthesesectionsthat
werenotapparent(ornotpresent)inthefrozensection.
Immunohistochemistry(immunocytochemistry)
Immunohistochemistryisaverypowerfultoolforidentifyingcellsandsubstancesintissuesectionsbyusingthespecificityofantigen
antibodybinding.Commerciallyavailableantibodiesareraisedagainstparticularcellularconstituentsandthese,whenappliedinsolution
tatissuesection,willbindspecificallytthoseconstituents.Theboundantibodyislinkedbyafurthersteptacolouredindicatorthatcanbe
seendownthemicroscope(staining).
Despitethespecificityofantigenantibodybinding,immunohistochemistryisnotaspecificscience.Stainingisingeneralreliableand
reproducible,particularlyusingthecommonestmarkers,forwhichavastamountofexperienceisnowavailable,butaberrantstainingcan
anddoesoccur(sometimesrelatedtvaryingfixation).Therefore,asinglepositiveornegativereactionshouldnotoverrulethebasic
histologicalfindingsinacase.Immunostainingshouldingeneralinvolveapanelofdifferentantibodiestminimisetheeffectofasingle
aberrantresult,andthepanelrequestedshouldbedesignedtansweraparticularquestionrelatedtthedifferentialdiagnosisbasedonthe
H&Eappearances.
Themajorusesofimmunohistochemistryaretidentifyapoorlydifferentiatedmalignanttumourasacarcinoma,sarcoma,melanomaor
lymphomatidentifyscantytumourcellsortumourobscuredbyinflammation(forinstanceassessingthedepthofaheavilyinflamed
melanoma)orforidentifyingadenselymphoidinfiltrateaslymphomabydemonstratingclonalityofimmunoglobulinlightchain
expression.Immunohistochemistryhaslittleroleinthediagnosisofinflammatoryskinconditions.
Themostcommonlyusedantibodiesarethoseraisedagainstcytoskeletalproteins(intermediatefilaments)suchascytokeratin,desmin
andactin,andmarkersofmelanocyticlineageordifferentiation.Therearealsnumerousmarkersoflymphoiddifferentiation.Hundredsof
markersarenowavailable(ofvariableutility!).Aselectionofthemostcommonlyusedonesisgivenbelow.SeeResourcesforalinkta
comprehensivelist.
AntibodyTarget

Antibody

Identifies

IntermediateFilaments

Cytokeratin
Desmin
Actin

Epithelialdifferentiation
Muscledifferentiation
Muscle(muchlessspecific)

Melanocyticdifferentiation

S100
HMB45
MelanA

Sensitive,butnonspecific
Morespecific,butnotsensitive
Morespecific,sensitive

Lymphoid/HaematologicalMarkers

Leucocytecommonantigen
CD3
CD4/CD8
CD20/CD79a
CD30
CD68
Kappa/Lambda

Mostmaturelymphocytes
Tcells
Tcellsubsets
Bcells
Largelymphoidcells
Macrophages
Immunoglobulinlightchains

Directimmunofluorescence

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Directimmunofluorescencestainingisaspecifictypeofimmunohistochemistry,usuallytdetectthepresenceofimmunoglobulinsand
complementintheskin.AbatchoffluoresceinisothiocyanatelabelledantibodiesagainstIgG,IgM,IgA,fibrinandC3isappliedtfrozen
sectionsoffreshtissueandexaminedbyfluorescencemicroscopy.
Characteristicstainingpatternsareseenintheimmunobullousdiseases,lupuserythematosusandvasculitis.
Circulatingskinantibodiesaredetectedbyindirectimmunofluorescence.Inthistechnique,thepatient'sserumisincubatedwitha
squamousepithelium,andanyantibodiesthatbindareidentifiedbyafluoresceinlabelledprobe.

Directimmunofluorescence

Normalskin

LinearbandofIgAatdermoepidermaljunction

Electronmicroscopy
Electronmicroscopy(EM)israrelyrequiredforroutinediagnosisandmanagementofskindiseases.ScanningEMisusefulfordiagnosing
raregenetichairshaftabnormalities.ScanningEMcanbeusedtidentifycertainmineraldeposits.ViralparticlescanbeidentifiedbyEM
e.g.,tconfirmadiagnosisoforf.
Itremainsausefultoolforresearchespeciallywiththerecentinterestinphotoageing,genetics,biotechnologyandmolecularbiology.
EMisbestperformedwhenfreshtissueisfixedwithglutaraldehyde.Suboptimal,butoccasionallyveryuseful,examinationcansometimes
bemadeonappropriatelytreatedformalinfixedtissueiffreshmaterialisnotavailable.Itcanbecombinedwithimmunohistochemistry
techniquesforworkingupinterestingcases,forthestudyofgeneticandautoimmunebullousdiseases.Itisavailablefromcertain
researchlaboratoriesinNewZealand.
Cytogenetics
Insomecasesidentifyingspecificchromosomalalterationscanconfirmadiagnosis.Thisisparticularlyusefulincutaneouslymphomas
whereitcanfurtherclassifythediagnosis,orhelpdifferentiatebetweensystemicorprimarycutaneousdisease.Thecommonlyused
techniqueisFISH(fluorescentinsituhybridisation)wherelabelledDNAprobesattachtknownareasonspecificchromosomes.Equally
immunohistochemistrycanbeusedtstainproteinsgainedorlostfromthesemutations.
Therecenttechniqueoflaserguidedtissuemicrodissectionisprovingusefulinidentifyinggeneexpressioninlocalisedareasoftissues
seenunderthemicroscope.DNAcanbeextractedfromdirectlyvisualisedclustersofcells.Singlenucleotidepolymorphism(SNP's)probes
asmarkersofknownmutationscanthenbeusedinconjunctionwithpolymerasechainreaction(PCR)tidentifywhetheraparticulargene
isbeingexpressedinthatareaoftheskinbiopsy.Thisislargelyaresearchtechniquecurrently.
Confocallaserscanningmicroscopy
Confocallaserscanningmicroscopyisusedforobtaininghighresolutionimagesand3Dreconstructionsinvivasitisapplieddirectly
tintactskintviewopticalslicesofthetissue.Thereisongoingactiveresearchwiththistechniqueindermatologyandtheremaybearole
forroutinediagnosticuseinthefuture.Ithasbeenusedtimagebenignandmalignantpigmentedskinlesions,nonmelanomaskin
cancer,inflammatoryskinconditions,anddynamicskinprocesses.Therehasalsbeensuccesswithmappingofindistinctpigmentedlesions
priortsurgery.

Activity
Examineaselectionofpathologicreports.Whatspecialstainshavebeenused,why,andwhatweretheresultsinthesecases?

Page3of6.Nexttopic:Inflammatorydermatoses.Backto:Dermatopathologycontents.
Relatedinformation
References:
Reference:MutasimDF,AdamsBB.Immunofluorescenceindermatology.JAmAcadDermatol2001Dec45(6):80322

OnDermNetNZ:
Dermatopathologyresources

Informationforpatients
Skinpathology

Otherwebsites:
FloridaStateUniversityWebPath
Electronmicroscopy
Specialstains

UniversityofNottinghamHistotechnologytechnicalmethods
StainsfileTheonlineresourceforhistotechnologists

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ArupAnatomicPathologyImmunohistochemistryStainOffering.
KanitakisJ.Immunohistochemistryoftheskin.EuropeanJournalofDermatology1998:853947

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Author:HonAssocProfAmandaOakley&DrPaulNewman.

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