Aperio Image Analysis
User’s Guide
Purdue University is an equal access/equal opportunity/affirmative action university.
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ii Aperio Image Analysis User’s Guide
Copyright©2008AperioTechnologies,Inc.
PartNumber/Revision:MAN‐0013,RevisionC
Date:September4,2009
ThisdocumentappliestosoftwareversionsRelease10.1andlater.
Allrightsreserved.Thisdocumentmaynotbecopiedinwholeorinpartorreproducedinanyothermediawithouttheexpress
writtenpermissionofAperioTechnologies,Inc.Pleasenotethatundercopyrightlaw,copyingincludestranslationintoanother
language.
User Resources
ForthelatestinformationonAperioTechnologiesproductsandservices,pleasevisittheAperioTechnologieswebsiteat:
http://www.aperio.com.
Disclaimers
UsenormalcareinmaintainingandusingtheSpectrumservers.InterruptingnetworkconnectionsorturningofftheSpectrumand
DSRserverswhiletheyareprocessingdata(suchaswhentheyareanalyzingdigitalslidesorgeneratinganauditreport)canresult
indataloss.
ThismanualisnotasubstituteforthedetailedoperatortrainingprovidedbyAperioTechnologies,Inc.,orforotheradvanced
instruction.AperioTechnologiesFieldRepresentativesshouldbecontactedimmediatelyforassistanceintheeventofany
instrumentmalfunction.InstallationofhardwareshouldonlybeperformedbyacertifiedAperioTechnologiesServiceEngineer.
ImageServerisintendedforusewiththeSVSfileformat(thenativeformatfordigitalslidescreatedbyscanningglassslideswith
theScanScopescanner).EducatorswilluseAperiosoftwaretoviewandmodifydigita lslidesinCompositeWebSlide(CWS)format.
AperioproductsareFDAclearedforspecificclinicalapplications,andareintendedforresearchuseforotherapplications.For
clearanceupdates,visitwww.aperio.com
Trademarks and Patents
ScanScopeisaregisteredtrademarkandImageServer,TMALab,ImageScope,andSpectrumaretrademarksofAperio
Technologies,Inc.Allothertradenamesandtrademarksarethepropertyoftheirrespectiveholders.
AperioproductsareprotectedbyU.S.Patents:6,711,283;6,917,696;7,035,478;7,116,440;7,428,324;7,457,446;7,463,761;7,502,519;
7,518,652;andlicensedunderoneormoreofthefollowingU.S.Patents:6,101,265;6,272,235;6,522,774;6,775,402;6,396,941;
6,674,881;6,226,392;6,404,906;6,674,884;and6,466,690.
Contact Information
Headquarters Europe Office Asia Office
Aperio Technologies, Inc.
1360 Park Center Drive
Vista, CA 92081
United States
Aperio UK Ltd
The Vineyard
Axbridge
Somerset BS26 2AN UK
Aperio Technologies, KK
UZ Building 5F
3-3-17, Surugadai
Kanda, Chiyoda-ku
Tokyo, Japan 101-0062
United States of America
Tel: 866-478-4111 (toll free)
Fax: 760-539-1116
Customer Service Tel: 866-478-4111 (toll free)
Technical Support Tel: 866-478-3999 (toll free)
Email Email: support@aperio.com
Europe
Tel: +44 (0) 1934 733679
Fax: +44(0) 1934 733660
Customer Service & Technical Support Tel: +44 (0) 1934 733679
Email Email: europesuppor[email protected]
Asia
Tel: +81-3-3259-5255
Fax: +81-3-3259-5256
Customer Service Email: asiainfo@aperio.com
Technical Support Email: asiasuppor[email protected]
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If you have trouble accessing this document because of a disability, please contact PVM Web Communications at [email protected].
Contents
CHAPTER 1 - INTRODUCTION ....................................................................... 1
AperioAlgorithms......................................................................................................1
Prerequisites ............................................................................................................2
FDAClearedImageAnalysisAlgorithms ............................................................. 2
IntendedUse ...............................................................................................................3
IHCQuickAnalysis ...................................................................................................3
Slide‐SpecificProcessing........................................................................................3
OtherOptions..........................................................................................................4
CHAPTER 2 - ALGORITHM INSTALLATION ......................................................... 7
WheretoInstalltheAlgorithm................................................................................7
CHAPTER 3 - OPENING A DIGITAL SLIDE FOR ANALYSIS ........................................ 9
AboutImageScope .....................................................................................................9
AccessingaDigitalSlideinSpectrum ................................................................. 10
OpeningaLocalDigitalSlide................................................................................12
OpeningaRemoteDigitalSlide............................................................................14
ForMoreInformation ..............................................................................................15
CHAPTER 4 - SELECTING AREAS TO ANALYZE .................................................. 17
CHAPTER 5 - REGISTERING THE ALGORITHM ON SPECTRUM ................................. 21
OpeningtheDigitalSlide.......................................................................................21
CreatingaNewMacro .............................................................................................22
SavingandRegisteringtheMacroonSpectrum ................................................26
TestingandModifyinganExistingMacro ..........................................................27
UsingtheTuningWindowtoTestAlgorithmParameters...............................28
CHAPTER 6 - RUNNING THE ANALYSIS........................................................... 31
AnalyzinginImageScope.......................................................................................31
SpectrumAnalysisBatchJobs................................................................................33
ForMoreInformation ..............................................................................................35
CHAPTER 7 - EXPORTING RESULTS ............................................................... 37
ExportingResultsasText........................................................................................38
ExportingResultstoaSpreadsheet....................................................................... 38
APPENDIX A - TROUBLESHOOTING ............................................................... 39
INDEX................................................................................................... 41
Aperio Image Analysis User’s Guide iii
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Contents
iv Aperio Image Analysis User’s Guide
Purdue University is an equal access/equal opportunity/affirmative action university.
If you have trouble accessing this document because of a disability, please contact PVM Web Communications at [email protected].
1 1
Introduction Introduction
This chapter introduces Aperio image analysis algorithms. This chapter introduces Aperio image analysis algorithms.
This user guide
discusses using
Aperio image
analysis algorithms
to analyze digital
slides.
Aperio's image
analysis algorithms
are FDA cleared for
specific clinical
applications, and are
intended for
research use for
other applications.
Please see the
Intended Use section
of the user guides
for the specific
cleared applications
you wish to use for
details on in vitro
diagnostic use.
Theprocessofanalyzingdigitalimagesbeginswiththedigitalslide,adigital
imagecreatedbyscanningglassslides.UsingAperioimageanalysisalgorithms
toanalyzedigitalslidesprovidesseveralbenefits:
Theprocessofanalyzingdigitalimagesbeginswiththedigitalslide,adigital
imagecreatedbyscanningglassslides.UsingAperioimageanalysisalgorithms
toanalyzedigitalslidesprovidesseveralbenefits:
Increasesproductivity–Imageanalysisautomatesrepetitivetasks. Increasesproductivity–Imageanalysisautomatesrepetitivetasks.
Improveshealthcare–Analyzingdigitalslideshelpsyoutoexamine
slidestainingtofindpatternsthatwilltellyoumoreabouttheslide.
Usinganalgorithmtolookforthesepatternsprovidesprecise,
quantitativedatathatisaccurateandrepeatable.
Improveshealthcare–Analyzingdigitalslideshelpsyoutoexamine
slidestainingtofindpatternsthatwilltellyoumoreabouttheslide.
Usinganalgorithmtolookforthesepatternsprovidesprecise,
quantitativedatathatisaccurateandrepeatable.
Developmentofnewcomputer‐basedmethods–Imageanalysishelps
youanswerquestionsthatarebeyondthecapabilitiesofmanual
microscopy,suchas“Whatisthesignificanceofmultiplestainsatthe
celllevelandcolocalizationofstains?”
Developmentofnewcomputer‐basedmethods–Imageanalysishelps
youanswerquestionsthatarebeyondthecapabilitiesofmanual
microscopy,suchas“Whatisthesignificanceofmultiplestainsatthe
celllevelandcolocalizationofstains?”
Workflowintegration–TheSpectrumdigitalpathologyinformation
managementsoftwaresuiteintegratesimageanalysisseamlesslyinto
yourdigitalpathologyworkflow,requiringnoadditionalworkbythe
laborpathologist.Withtheclickofabutton,thealgorithmisexecuted
whileyoureviewthedigitalslide.
Workflowintegration–TheSpectrumdigitalpathologyinformation
managementsoftwaresuiteintegratesimageanalysisseamlesslyinto
yourdigitalpathologyworkflow,requiringnoadditionalworkbythe
laborpathologist.Withtheclickofabutton,thealgorithmisexecuted
whileyoureviewthedigitalslide.
Aperio Algorithms Aperio Algorithms
Analyzingadigitalslideisdonebyapplyingalgorithmsdirectlytothedigital
slideorselectedregionsofthedigitalslideafteropeningthedigitalslidein
ImageScope.
Analyzingadigitalslideisdonebyapplyingalgorithmsdirectlytothedigital
slideorselectedregionsofthedigitalslideafteropeningthedigitalslidein
ImageScope.
Usingimageanalysisalgorithmscananswersuchquestionsas:Usingimageanalysisalgorithmscananswersuchquestionsas:
Whereandhowmuchstainingisthere? Whereandhowmuchstainingisthere?
Whereandhowmanyobjects(forexample,tumorcells)arethere? Whereandhowmanyobjects(forexample,tumorcells)arethere?
Howmuchstainingisthereondifferentobjects(forexample,cell
nuclei)?
Howmuchstainingisthereondifferentobjects(forexample,cell
nuclei)?
Severalalgorithmsdevelopedforspecificapplicationsareavailable,including:Severalalgorithmsdevelopedforspecificapplicationsareavailable,including:
PositivePixelCount–Thisalgorithmquantifiestheareaandintensities
oftwostainsbasedonuser‐definedvaluesforcolorandintensity
thresholds.
PositivePixelCount–Thisalgorithmquantifiestheareaandintensities
oftwostainsbasedonuser‐definedvaluesforcolorandintensity
thresholds.
Aperio Image Analysis User’s Guide 1
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Chapter 1 – Introduction
ColorDeconvolution–Theprofessionalversionofthepositivepixel
countalgorithm,ColorDeconvolutionaccuratelyseparatesstains,
resolvingmulti‐staincolocalizationproblems.Thisalgorithmseparates
theimageintothreechannelscorrespondingtotheactualcolorsofthe
stainsused.Thisallowsthepathologisttoaccuratelymeasurethearea
foreachstainseparately,evenwhenthestainsaresuperimposedatthe
samelocation.
Colocalization–TheColocalizationalgorithmisanextensionofthe
ColorDeconvolutionalgorithm,quantifyingtheareaandintensitiesof
“colocalized”markers.
RareEventDetection–Therareeventdetectionalgorithmisoftenused
todetectmicrometastasisoftumorcellsfoundincirculatingblood.
ThePositivePixelCountalgorithmisprovidedforfreewithAperiosoftware.
OtheralgorithmsareavailablefromAperioforafee.Algorithmshavealsobeen
developedbythirdpartiesandtoolsareavailablefromAperioforcreatingyour
ownalgorithms—contactAperiofordetails.
Thesealgorithmsallhavecontrolparameters—forexample,intensityandhue
settings—thatallowthealgorithmtobetailoredtoyourspecificneeds.
Prerequisites
TheexamplesinthisguideassumeyouareusingAperioRelease10orlater.
BecauseAperiodigitalslidesarebydesignhighresolutionandinformationrich,
forbestresultsyoushoulduseahighqualitymonitortoviewthem.Makesure
themonitorisattheproperviewingheightandinaroomwithappropriate
lighting.WerecommendanyhighqualityLCDmonitormeetingthefollowing
requirements:
Display Type: CRT minimum, LCD (flat panel) recommended
Screen Resolution: 1024(h) x 768(v) pixels minimum, 1920 x 1050 or larger
recommended.
Screen Size: 15” minimum, 19” or larger recommended
Color Depth: 24 bit
Brightness: 300 cd/m
2
minimum, 500 or higher recommended
Contrast Ratio: 500:1 minimum, 1000:1 or higher recommended
FDA Cleared Image Analysis Algorithms
SeveralAperioalgorithmshavebeenclearedbytheFDAforclinicalusewhen
usedonScanScopemodelsthatarelabeledasapprovedmedicaldevices,andare
intendedforresearchuseforotherapplications.Thesealgorithmshavetheirown
userguides.PleaseseetheIntendedUsesectionoftheuserguidesforthe
specificclearedapplicationsyouwishtousefordetailsoninvitrodiagnostic
use.
2 Aperio Image Analysis User’s Guide
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Chapter 1 – Introduction
Intended Use
Algorithmsareintendedtobeusedbytrainedpathologistswhohavean
understandingoftheconditionstheyaretestingforinrunningthealgorithm
analysis.
Eachalgorithmhasinputparametersthatmustbeadjustedbyanexpertuser
whounderstandsthegoalofrunningtheanalysisandcanevaluatethealgorithm
performanceinmeetingthatgoal.
Youwilladjust(tune)theparametersuntilthealgorithmresultsaresufficiently
accurateforthepurposeforwhichyouintendtousethealgorithm.Youwill
wanttotestthealgorithmonavarietyofimagessoitsperformancecanbe
evaluatedacrossthefullspectrumofexpectedimagingconditions.Tobe
successful,itisusuallynecessarytolimitthefieldofapplicationtoaparticular
tissuetypeandaspecifichistologicalpreparation.Amorenarrowlydefined
applicationandconsistencyinslidepreparationgenerallyequatestoahigher
probabilityofsuccessinobtainingsatisfactoryalgorithmresults.
Ifyougetalgorithmanalysisresultsthatarenotwhatyouexpected,pleasesee
AppendixA,“Troubleshooting”onpage39forassistance.
IHC Quick Analysis
TheexamplesinthisguideshowtheuseoftheImageScopedetailedAnnotations
window,whichprovidesageneralsolutionforimageanalysis.However,amore
streamlinedversionofimageanalysisisalsoavailablethatusestheImageScope
Annotationssummaryview.TheAnnotationssummaryviewwasspecifically
developedforanalyzingIHCdigitalslidesandmaketheprocessquickerand
simplerbyfittingintoapathologist’sorresearcher’snormalactivities.
FordetailsonusingtheDigitalIHCfeatures,onsetupstepsforSpectrum,and
forguidelinesoncreatingvalidationstudiesforthespecificIHCapplicationyou
wanttouse,see:
DigitalIHCUserʹsGuide
DigitalIHCGuidetoSpectrumSetup
TheuserʹsguideforthespecificIHCapplicationyouareusing.
Slide-Specific Processing
ThekeytotheDigitalIHCworkflowisslide‐specificprocessing,whichdefineshow
adigitalslidewillbeprocessedbasedonitsstainandtypeoftissue(bodysite).
Theslide‐specificprocessingcandefinewhatalgorithmwillbeusedtoanalyze
thattypeofslide,howanalysisresultswillbedisplayandhowtointerpretthose
results(alternatively,manualscoringcanbesetupfortheslide),andwhat
commentswillbeavailabletobeusedbythepathologistorresearcherviewing
theslide.
Aperio Image Analysis User’s Guide 3
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Chapter 1 – Introduction
Theslide‐specificconfigurationforeachstain/bodysitecombinationisdefined
bytheSpectrumadministrator.SeetheDigitalIHCGuidetoSpectrumSetupfor
details.
Onceslide‐specificprocessingissetup,viewing,annotating,andanalyzinga
digitalslidebecomesaquickprocessthattakesjustafewmouseclicks.
ThesummaryviewoftheAnnotationswindowisdesignedspecificallyfor
workingwithIHCdigitalslidestoprovideaquickwaytomarktumorregions
andanalyzetheminonesimplestep.
ToopentheAnnotationswindowinsummaryview:
1. IdentifyadigitalslideinSpectrumforwhichstain/bodysiteslide‐
specificprocessinghasbeendefined.
2. FromtheSpectrumpage,openthedigitalslideinImageScopeby
clickingitsthumbnail.TheAnnotationswindowinsummaryview
appears.(Ifthewindowdoesnotlooklikethis,clicktheSummary
buttontoreturntheAnnotationswindowtothesummaryview):
Thealgorithmappropriateforthistypeofslideislistedinthedrop‐downbox.
Youcanselectanotheralgorithmifyouwishfromthatbox.
Fromthiswindowyoucandrawannotationstoidentifyareastoanalyzeandrun
theanalysisallinoneeasystep.
FordetailsonusingtheAnnotationssummaryviewwindow,seetheDigitalIHC
User’sGuide.
Other Options
Tonavigatebetweenannotationsyouhavedrawn,usethenumbered
buttonsorarrowkeys.(Asyoudrawannotations,thebuttonsatthetop
ofthewindowdisplayanumberforeachannotation.)Whenyouselect
anannotationinthisway,thatannotationiscenteredintheImageScope
window.
4 Aperio Image Analysis User’s Guide
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Chapter 1 – Introduction
Toseeareportimageyouhaveselected,selectReportImagefromthe
drop‐downlist.
Tocreateanannotationthatwillnotbeusedforanalysis(forexample,a
rulerorarrow),selectAnnotationsfromthedrop‐downlistbefore
drawing.
Aperio Image Analysis User’s Guide 5
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Chapter 1 – Introduction
6 Aperio Image Analysis User’s Guide
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2
Algorithm Installation
The instructions in this section apply to all Aperio
algorithms.
IfyoupurchasedanalgorithmwhenyoupurchasedaScanScope,thealgorithm
mayalreadybeinstalledonyourScanScopeserver.(Inthiscase,thealgorithm
installerprogramwillalsobelocatedonyourScanScopeserverin
C:\AperioInstall).
Or,youmayhavereceivedinformationfromTechSupportonwhereto
downloadthealgorithminstallersoyoucaninstallthealgorithmyourself.
InstallinganalgorithmisasimplematterofusingWindowsExplorertonavigate
tothelocationofthealgorithminstallerfileanddouble‐clickingthefilename.
Followtheinstructionsofthealgorithminstaller,clickingNextwheninstructed
todoso.
Where to Install the Algorithm
Whereyouinstallthealgorithmdependsonhowyouaregoingtobeanalyzing
digitalslides:
AnalyzingdigitalslidesthroughSpectrum–Ifyouaregoingtobe
usingSpectrumtoanalyzeasingledigitalslideorabatchofdigital
slides,youwillneedtoinstallthealgorithmonbothyourlocal
workstationandonDSR(DigitalSlideRepository),whichistheserveror
computersconnectedtotheserveronwhichAAFClientisinstalled.
YouwillalsoneedtoregisterthealgorithmmacroonSpectrum.See
Chapter5,“RegisteringtheAlgorithmonSpectrum”onpage21for
details.
Analyzinglocaldigitalslides–Ifyouwillbeusingthealgorithmto
analyzelocaldigitalslides(thatis,digitalslidesthatarelocatedonyour
workstationoronyournetworkwheretheycanbeaccessedbyMicrosoft
filesharing),installthealgorithmonyourworkstation.However,be
awarethatwhenanalyzinglocaldigitalslides,theanalysisresultsare
storedonyourworkstation,notintheSpectrumdatabase.
Analyzingremotedigitalslides–Ifyouwillbeusingthealgorithmto
analyzeremotedigitalslides(thatis,digitalslidesthatarelocatedon
yourAperioImageServer)byopeningthedigitalslidedirectlyonthe
Aperio Image Analysis User’s Guide 7
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Chapter 2 – Algorithm Installation
8 Aperio Image Analysis User’s Guide
ImageServer,installthealgorithmontheDSR(theserverorcomputers
connectedtotheserveronwhichAAFClientisinstalled).
Creatinganalysismacros–Nomatterwhereyouaregoingtoanalyze
digitalslides,ifyouaregoingtocreateanalgorithmmacro,youneedto
installthealgorithmonyourlocalworkstationaswellasonthelocations
discussedabove.(Youwillneedtocreateandsaveanalgorithmmacro
toanalyzedigitalslidesonSpectrum.)
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3
Opening a Digital Slide for
Analysis
Before you can analyze a digital slide, you will open it for
viewing in ImageScope. You can either open the slide in
Spectrum or directly from ImageScope.
Thereareseveraldifferentwaystoopenadigitalslideforviewingdependingon
whetherthedigitalslideislocalorremote.
Localdigitalslidesresideonyourworkstationoronyourlocalnetwork
whereyoucanaccesstheminImageScopeusingMicrosoftfilesharing.
Remotedigitalslidesresideonaremoteserverwhereyoucanaccess
themdirectlyinImageScopeorbyfirstgoingthroughSpectrum.
Inmostcases,weexpectyoutoopenadigitalslideinSpectrum.Othermethods
ofaccessarediscussedattheendofthischapter.
About ImageScope
InmostcasesyouwillbeusingImageScopetoviewandanalyzedigitalslides.In
additiontothebasicfeaturesshowninthisguide,ImageScopealsocontains
manyadvancedfeaturesthatwillhelpyouviewandassessdigitalslides:
ImageQuality(IQ)–theIQfeatureisavailablewhenyoursiteis
licensedforSpectrumPlusandwhenyouopenadigitalslidein
ImageScopefromSpectrum.IQcontainsavarietyoftoolsthathelpyou
tobetterassessadigitalslidebasedonitsstain.Inadditiontoallowing
youtoviewstainchannelsseparately,IQalsoremapsstaincolors,
accentuatescellulardetailandrecombinesstains.Fordetails,seetheIQ
ImageQualityUser’sGuide.
ScaleAxesandGrid–Youcanoptionallyoverlaythedigitalslideimage
withmeasurementaxesandaxes/grid.
ImageScopeRuler–TheImageScoperulertoolallowsyoutomeasure
anobjectonthedigitalslidebydrawingontheslide.
Formoreinformation,seetheImageScopeUser’sGuide.
Aperio Image Analysis User’s Guide 9
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Chapter 3 – Opening a Digital Slide for Analysis
Accessing a Digital Slide in Spectrum
Cases(orprojects),specimensanddigitalslidesaremanagedusingAperio’s
Spectrum.Apathologistwhowantstoaccessadigitalslidefirstneedstologinto
Spectrumandnavigatetothecaseandthespecimenthatshowsthelistofits
associateddigitalslides.
To analyze digital
slides and to create
and change
algorithm macros,
the user role you log
in with must have
the following
command
permissions
enabled:
Analysis Macros –
Add, Edit
Images – Analyze.
Contact the
Spectrum
administrator if you
are not sure if your
user role allows
these actions.
PleaseaskyourSpectrumadministratorfortheSpectrumweb‐address,your
logonname,andapassword.
1. OpenMicrosoftInternetExplorer(oranotherwebviewerlikeFirefox)
andentertheSpectrumweb‐address.Youseealoginpage.
2. LogontoSpectrumbyenteringyourusernameandpassword.(The
passwordiscasesensitive.)
3. Ifmorethanoneuserroleisassignedtoyourlogin,youareaskedto
choosewhichuserroleyouwanttologinwith:
10 Aperio Image Analysis User’s Guide
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Chapter 3 – Opening a Digital Slide for Analysis
Afterloggingin,youseetheSpectrummainpage:
TheexactcategoriesofdatayouseedependonyourSpectrum
configurationandoptions.Forthepurposesofthisguide,wewill
assumeSpectrumPlusisconfiguredforclinicalorresearchuse.
4. NavigatetothelistofcasesbyclickingListallCases(ortothelistof
projectsbyclickingListallProjects).
Aperio Image Analysis User’s Guide 11
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Chapter 3 – Opening a Digital Slide for Analysis
5. Openthecaseorprojectyouwanttoreviewbyclickingtheopendata
icon
forthecaseorproject.
Younowseethecaseorprojectinformationpagethatcontainsdetails
aboutthatcaseorproject.
6. Onthedetailpage,movedowntotheDigitalSlidesareaofthepageand
openadigitalslideinImageScopebyclickingitsthumbnailinthedigital
slidelist.
Althoughyouusuallywillwanttofindadigitalslidebygoingtoitsparentcase,
project,orspecimen,youcanalsouseSpectrum’ssearchfunctionstofinda
specificdigitalslide,oryoucangotothedigitalslidetableonthemainpageto
browsealldigitalslides.
Opening a Local Digital Slide
Toopenadigitalslidethatresidesonyourworkstationorlocalareanetwork:
1. StartImageScopebyclickingStart,pointingtoAllPrograms>
ScanScope,andthenselectingImageScope.
2. GototheFilemenuandselectOpenImage(orclick
onthe
ImageScopetoolbar).
12 Aperio Image Analysis User’s Guide
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Chapter 3 – Opening a Digital Slide for Analysis
3. OntheOpenImagewindow,navigatetothelocationthatcontainsthe
imageyouwanttoview.
4. ClickthenameofthedigitalslideyouwanttoopenandclickOpen.
YoumayneedtochangethefiletypeintheOpenImagewindowtoseethetype
ofimageyouwanttoview.Forexample,toviewaCWSimage,clickthefiletype
drop‐downlistandselectCompositeWebSlides(*/SlideScan.ini).
Aperio Image Analysis User’s Guide 13
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Chapter 3 – Opening a Digital Slide for Analysis
Opening a Remote Digital Slide
ToopenaremotedigitalslidedirectlyfromwithinImageScopewithoutgoing
throughtheSpectrumuserinterface:
1. GototheImageScopeFilemenuandselectAccessRemoteServerto
connecttoSpectrum.Enterthenameoftheservercontainingthedigital
slides(usuallytheserveronwhichSpectrumresides):
2. SetthePortvalueto82.
3. ClickConnect.
4. Whenaskedforyourusernameandpassword,enteryourSpectrum
usernameandpassword.
14 Aperio Image Analysis User’s Guide
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Chapter 3 – Opening a Digital Slide for Analysis
5. NowyouseeapageofdigitalslidesontheSpectrumsite.Youcan
choosebetweentwoviews:ListandThumbnailbyselectingtheview
youwantfromthedrop‐downlistattheupperright.
6. SelectadigitalslidebyclickingImageScopebeneathanimage.
For More Information
FormoreinformationonopeningadigitalslideinImageScope,seethe
ImageScopeUser’sGuide.
Aperio Image Analysis User’s Guide 15
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Chapter 3 – Opening a Digital Slide for Analysis
16 Aperio Image Analysis User’s Guide
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4
Selecting Areas to Analyze
You can analyze an entire digital slide or just selected areas.
This chapter discusses how to select areas of the digital
slide to analyze.
WhetheryoudecidetouseanalgorithmdirectlyinImageScopeorwishtouse
Spectrum’sbatchanalysisfeature,youmaywanttofirstuseImageScopetoselect
areasofinterestinthedigitalslidetoanalyze.
1. OpenadigitalslideinImageScope.SeeChapter3“OpeningaDigital
SlideforAnalysis”onpage9forinstructionsonopeningadigitalslide
inImageScope.
Afteropeningthedigitalslide,youseethemainImageScopeviewing
window:
Aperio Image Analysis User’s Guide 17
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Chapter 4 – Selecting Areas to Analyze
2. NowyoucanusetheImageScopedrawingtoolstoselect(orexclude)
areastoanalyze.
Forexample,intheimagebelowweusedthefollowingtoolsonthe
ImageScopetoolbar:
Freehand pen – Use to draw a free-form area of interest.
Negative freehand pen – Use to draw an area to
exclude
from the
analysis. Note that you can use this in combination with the other
drawing tools to first select an area of interest and then exclude areas
within the selected area that you do not want to analyze.
Rectangle tool – Draws a rectangular area. If you want to select a
square, hold down the Shift key while drawing.
Ifyouhavedefinedafixedsizeforannotations,holddowntheControl
keywhileyouusethedrawingtooltodrawanannotationofthat
predefinedsize.(SeetheImageScopeUser’sGuidefordetailsonthis
feature.)
Freehand
negative pen
Freehand
negative pen
Rectangle
tool
3. Toseeinformationabouttheannotationsyouhavedrawn,gotothe
ImageScopeViewmenuandselectAnnotations.
18 Aperio Image Analysis User’s Guide
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Chapter 4 – Selecting Areas to Analyze
EachofthethreeannotationswedrewislistedintheLayerRegions
panel,withareaandlengthmeasurements(inmicrons).Toselecta
specificannotation(forexampleifyouwanttodeleteitormoveit),click
itintheImageScopeimageorclickitintheLayerRegionspanelofthe
AnnotationswindowandyouseeithighlightedontheImageScopemain
window.
Forexample,belowweclickedonannotation1(therectangular
annotation)andseethatitsboundarycontainsadarklineshowingitis
selected:
Todeletetheselectedannotation,presstheDeletekeyonyourkeyboard
orclicktheredXintheLayerRegionspane.Tomoveit,holddownthe
ControlkeyanddragitontheImageScopemainwindow.
Aperio Image Analysis User’s Guide 19
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Chapter 4 – Selecting Areas to Analyze
20 Aperio Image Analysis User’s Guide
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5
Registering the Algorithm on
Spectrum
An algorithm macro is an algorithm and its settings. Before
you can use an algorithm on Spectrum, you must create an
algorithm macro and save it on Spectrum.
ToanalyzeadigitalslideonSpectrum,youwillneedtocreateanalgorithm
macroandregisteritonSpectrum.Creatingamacroisasimplematterof
adjustingtheparametersofthealgorithmandsavingthosesettings.
Inordertocreateanalgorithmmacro,youneedtoinstallthealgorithmonboth
yourlocalworkstationandonSpectrum.(Thisconsistsofsimplyrunningthe
algorithminstalleronbothcomputers.)
NotethatcreatingandregisteringamacrorequiresthatyoulogintoSpectrumas
anadministrator.
Opening the Digital Slide
YoucaneitheropenaremotedigitalslideinSpectrumoropentheremotedigital
slidedirectlyinImageScope.SeeChapter3,“OpeningaDigitalSlidefor
Analysis”onpage9fordetails.
Openadigitalslideyouwillusetocreateanalgorithmmacro.
Aperio Image Analysis User’s Guide 21
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Chapter 5 – Registering the Algorithm on Spectrum
Creating a New Macro
1. LogintoSpectrumasanadministrator.
2. FromSpectrum,afteropeningadigitalslideinImageScope,gotothe
ImageScopeViewmenuandselectAnalysis.YouseetheAlgorithm
ServerJobwindow:
Becauseyouaregoingtocreateanewalgorithmmacro,youwon’tselect
oneofthemacroslistedinthewindow.
3. IntheRegionofAnalysissection,clickoneofthefollowingselections:
a) EntireImage–Whenanalyzingtheimage,testinganexistingmacro,
orcreatinganewmacro,theentireimagewillbeused.
b) SelectedAnnotation–Whenanalyzingtheimage,testingan
existingmacro,orcreatinganewmacro,onlytheportionofthe
imagedefinedbytheactiveannotationwillbeused.Ifno
annotationsexistfortheimage,EntireImageisautomatically
selected.
22 Aperio Image Analysis User’s Guide
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Chapter 5 – Registering the Algorithm on Spectrum
4. ClicktheCreatebuttontomakeanewmacro.TheSelectanAlgorithm
windowappears:
5. Selectthealgorithmyouwanttocreateamacroforbyclickingitinthe
listandthenclickingSelect.Ifyoudon’tseethealgorithmyouwantto
uselisted,youwillneedtoinstallthatalgorithmontheSpectrumserver
andonyourworkstation.
TheCreatebuttoncreatesanewmacrobasedontheselectedalgorithm.
Thisloadsthealgorithmmacrowithitsdefaultparameterssoyoucan
seeitsunmodifiedparameters.TheAnalysiswindownowappears.
6. Ifnotalreadyselected,clickInputsandmodifytheparameters(referring
totheuserguideforparticularalgorithmyouareusingforguidelines).If
Aperio Image Analysis User’s Guide 23
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Chapter 5 – Registering the Algorithm on Spectrum
thealgorithmyouareusingdoesnothaveanyinputs,skiptothenext
step.
7. ClickOutputsandselecttheoutputresultsyouwanttodisplayin
Spectrum(clearingthecheckboxesnexttotheresultsyoudon’twantto
display):
8. ClickRuntotestthemacroonthedigitalslide.
Instead of clicking
Run, you can click
the Tune button to
open a tuning
window in which you
can see instant
feedback on
parameter changes.
See “Using the
Tuning Window to
Test Algorithm
Parameters” at the
end of this chapter
for details.
Whentheanalysisiscomplete,themarkupimageinthemainImageScope
windowshowstheresults:
Inthisexample,wedrewarectangletoselecttheareawewantedtoanalyze,but
alsousedthenegativefreeformpentoexcludeareaswithintherectanglefrom
analysis.(SeeChapter4,“SelectingAreastoAnalyze,”onpage17for
informationonusingtheImageScopedrawingtools.)
24 Aperio Image Analysis User’s Guide
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Chapter 5 – Registering the Algorithm on Spectrum
Forinformationonthemeaningofthecolorsshowninthemark‐upimage,see
thenumericalresultsdisplayedintheAnnotationswindowandthe
documentationforthealgorithmyouareusing.
Toseethenumericalresultsoftheanalysis:
1. GototheImageScopeViewmenuandselectAnnotations.
Notethecolorshownnexttoeachparameter—thatisthecolorforthatresult
showninthemark‐upimage.
Keepadjustingthealgorithmparametersuntilyouaresatisfiedwiththeresults.
Thensavethemacro(seethenextsection).
Aperio Image Analysis User’s Guide 25
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Chapter 5 – Registering the Algorithm on Spectrum
Saving and Registering the Macro on Spectrum
Afteryouhavecreatedthemacro,saveittoregisteritonSpectrum.
1. OntheAnalysiswindow,clickSaveMacrotosavethemacroand
registeritonSpectrum:
2. Youareaskedtoenteranameforthemacro:
Typeanamethatwillhelpyouidentifythemacrointhefutureandclick
OK.
Younowseeamessagelettingyouknowthatthemacroissaved.Itis
nowregisteredonSpectrum.
Now,whenyoubringuptheAnalysiswindow,youwillbeabletoselectyour
macrobyclickingSelectAlgorithmbecauseyourmacrowillappearinthelist.
26 Aperio Image Analysis User’s Guide
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Chapter 5 – Registering the Algorithm on Spectrum
Testing and Modifying an Existing Macro
The Test and Create
buttons are disabled
if you are not logged
in as a Spectrum
administrator as
only administrators
can create and
modify algorithm
macros.
The Analyze button
is disabled if your
user permissions are
not set to Full
Control for the data
group containing the
digital slide image or
if your user role
does not permit
image analysis.
AfteropeninganimagefromSpectrum,whenyouselectAnalysisfromtheView
menu,youseetheAlgorithmServerJobwindow:
TheTestbuttonmodifiesanexistingmacroforthealgorithmyouhaveselected
andtestsitbeforesavingittoSpectrum.UsingtheTestbuttonloadsthe
algorithmmacrowithitsexistingparameters.
Aperio Image Analysis User’s Guide 27
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Chapter 5 – Registering the Algorithm on Spectrum
ModifytheparametersandclickRuntotestitonthedigitalslide.(Orclickthe
Tunebuttontotestthealgorithmparametersasdiscussedinthenextsection.)
Whenyouaresatisfiedwiththeresults,clicktheSaveMacrobuttonasdiscussed
intheprevioussectiontosavethemodifiedmacro.
Using the Tuning Window to Test Algorithm
Parameters
Thealgorithmtuningwindowgivesyouawaytoquicklyseetheresultsof
analyzingadifferentareaofanimageortotestchangesyoumaketothe
algorithmparameters.
These instructions
discuss using the
tuning window when
opening an image
from Spectrum.
You can also use the
algorithm tuning
window when you
open a local image—
just remember that
in this case analysis
results will be saved
locally, not in the
Spectrum database.
For details on
analyzing local
images, see the
ImageScope User’s
Guide
.
Tousethealgorithmtuningwindow:
1. InSpectrum,openadigitalslideinImageScope.
2. InImageScope,gototheViewmenuandselectAnnotationstoopenthe
Annotationswindow.Thiswindowiswhereyournumericalgorithm
analysisresultsappear.
3. GototheViewmenuandselectAnalysis.
YouseetheAlgorithmServerJobwindowwhereyoucanselectthe
algorithmmacroyouwanttouse.
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Chapter 5 – Registering the Algorithm on Spectrum
4. SelectthemacroyouwanttouseandclickTest.Youseethealgorithm
parameterwindow.
5. Onthealgorithmparameterwindow,clickTune.OntheImageScope
mainwindow,youseeanewAlgorithmTuningwindowwiththemark‐
upimagefromtheanalysisusingthecurrentparameters.
Aperio Image Analysis User’s Guide 29
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Chapter 5 – Registering the Algorithm on Spectrum
30 Aperio Image Analysis User’s Guide
YouseethenumericresultsoftheanalysisintheAnnotationswindow:
6. ToadjustthesizeoftheAlgorithmTuningwindow,grabacorneruntil
youseethedouble‐headedarrowandpullthewindowtochangethe
size.
7. Toseetheanalysisofanotherareaofthedigitalslide,dragthe
AlgorithmTuningwindowtoanotherareaormovethedigitalslide
underthewindow.
Every time the
Algorithm Tuning
window updates the
analysis, a new
mark-up image
appears in the
window and the
numeric data in the
Annotations window
changes to reflect
the new analysis.
8. Toseetheresultsoftheanalysiswhenyouchangetheparameters,
simplychangetheparametersinthealgorithmparameterswindowand
theAlgorithmTuningwindowupdatestoshowtheanalysisusingthe
newparameters.
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6
Running the Analysis
This chapter discusses how to use an algorithm in Spectrum
to analyze a digital slide. This is a general discussion which
applies to all algorithms. For details on using a specific
algorithm, refer to the user guide for that algorithm.
Onceyouhaveidentifiedthedigitalslideorslidesyouwanttoanalyze,youcan
analyzethemtogetherinaSpectrumbatchanalysis,oryoucanopeneachonein
ImageScopeandanalyzethedigitalslidesoneatatime.
Analyzing in ImageScope
Youcaneitheranalyzetheentiredigitalslideoronlyselectedportionsofit(see
theprevioussectionforinformationonselectedareasofanalysis).
ToanalyzeadigitalslidedirectlyinImageScope:
1. OpenadigitalslideinImageScope.See“AccessingaDigitalSlidein
Spectrum”onpage10forinstructionsonopeningadigitalslidein
ImageScopefromwithinSpectrum.Fordetailsonopeningadigitalslide
thatresidesonyourworkstationoronanon‐Spectrumremoteserver,
see“OpeningaLocalDigitalSlide”onpage12and“OpeningaRemote
DigitalSlide”onpage14.
Forexample(inthiscasewehaveselectedareastoanalyzeasdiscussed
intheprevioussection):
Aperio Image Analysis User’s Guide 31
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Chapter 6 – Running the Analysis
2. GototheViewmenuandselectAnalysis.TheAlgorithmServer
windowappears:
3. Clickanalgorithmmacrotoselectit.(Ifthealgorithmyouwanttouse
doesn’tappearinthiswindow,amacroforthealgorithmhastobe
createdandsavedonSpectrum.Seethepreviouschapterfordetails.)
4. Clicktheappropriateradiobuttontoselecttheareaofthedigitalslideto
analyze:
a) EntireImage–ThepartoftheimageshownintheImageScopemain
window.
b) SelectedAnnotation–Analyzeonlytheareasselectedbydrawn
annotationsinthecurrentannotationlayer.(Ifyouhavemorethan
onelayerofannotations,gototheAnnotationswindowandselect
theannotationlayerintheLayerspanethatcontainsthedrawings
thatselecttheareastoanalyze.)
5. SelecttheGenerateMarkupImagecheckboxtocreateapseudo‐color
markupimage.
32 Aperio Image Analysis User’s Guide
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Chapter 6 – Running the Analysis
7. ClickAnalyzetostarttheanalysis.IfyouhaveselectedtheGenerate
MarkupImagecheckbox,theimageintheImageScopewindowshows
thevisualresultsoftheanalysis.TheAnnotationswindowshowsthe
analysisresultdata.YoucanexporttheanalysisresultsinanExcel
spreadsheetorastextusingtheiconsintheAnnotationswindow(see
Chapter7,“ExportingResults,”onpage37fordetails).
Spectrum Analysis Batch Jobs
Spectrumprovidesaconvenienttoolforbatchanalysisofslides.Beforeyoucan
batchanalyzeselectedslideimagesinSpectrum,theSpectrumadministrator
mustfirstcreateamacroforeachimageanalysisalgorithmyouwanttousefor
analysis.
AnalysiscanbetakingplacewhileyoucontinuetoworkwithinSpectrum,
organizingdataandannotatingimages.
1. Displaythedesiredlistofdigitalslides.
Openaspecificcaseorprojecttoseethedigitalslidesassociated
withthatcase/project.
YoumaylistallslidesbygoingtotheDigitalSlidesmenuand
selectingAllDigitalSlides(AsList).
Or,youmaywanttosearchwithintheslidesforaspecificstainor
status.(SeetheSpectrum/SpectrumPlusOperator’sGuideformore
information.)
Aperio Image Analysis User’s Guide 33
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Chapter 6 – Running the Analysis
2. Selecttheboxtotheleftofeachslidetobebatchanalyzed.
3. ClickAnalyze.
TheAnalysispageappears:
The list of
annotation layers
you see in the Select
Input Annotation
Layer section of the
Analysis page
depends on the
digital slides you
have selected to
batch analyze.
The number of
annotation layers all
the selected slides
have in common are
listed in this section.
This is because
Spectrum does not
allow you to select
an annotation layer
that does not exist
for all the selected
digital slides. (In this
example, all slides
have three or more
annotation layers.)
If one of the digital
slides selected has
zero annotation
layers, you will only
see Most Recent or
Whole Image as
selections.
4. Selecttheanalysismacroofyourchoice.Ifyoudon’tseethealgorithm
youwanttouse,seeChapter5,“RegisteringanAlgorithmonSpectrum”
onpage21forinstructionsoncreatingandsavinganalgorithmmacro.
5. Ifyouwanttoanalyzeonlyaportionoftheimage,selectaspecific
annotationlayerintheSelectInputAnnotationLayersectionorclick
MostRecenttoselecttheannotationsinthemostrecentlycreated
annotationlayer.Ifyouwanttoanalyzetheentiredigitalslide,select
WholeImage.
(SeeChapter4,“SelectingAreastoAnalyze,”onpage17forinformation
ondrawingannotationstoselectareastoanalyze.)
6. SelecttheCreatemarkupimage(s)checkboxifdesired.
34 Aperio Image Analysis User’s Guide
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Chapter 6 – Running the Analysis
7. ClickAnalyze.Theslideschosenforanalysiswillbeaddedtothe
AnalysisJobQueue.
SeetheSpectrum/SpectrumPlusOperator’sGuideforinformationon:
Checkingtheprogressoftheanalyses.
Cancelinganalysisjobs.
Viewingtheanalysishistoryforspecificdigitalslides.
For More Information
FordetailsonusingAperioalgorithmstoanalyzedigitalslides,see:
Spectrum/SpectrumPlusOperator’sGuideforinformationonusing
algorithmsto analyzeindividualdigitalslidesorbatchesofdigitalslides.
ImageScopeUser’sGuideforinformationonusingalgorithmstoanalyze
localdigitalslidesonyourworkstationorlocalnetwork,ortoanalyze
remotedigitalslidesonanImageServer.
Aperio Image Analysis User’s Guide 35
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Chapter 6 – Running the Analysis
36 Aperio Image Analysis User’s Guide
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7
Exporting Results
This chapter discusses how to export analysis results as
text or Excel spreadsheet files.
Algorithmanalysisresultsarestoredinanannotationlayersoyoumaywantto
exportthatinformationintoatextfiletoincludeitinareportortochartthe
informationinaspreadsheetprogram.
Toseethenumericalresultsoftheanalysis:
1. GototheImageScopeViewmenuandselectAnnotations.The
Annotationswindowappearswiththeresultsdisplayed:
Aperio Image Analysis User’s Guide 37
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Chapter 7 – Exporting Results
38 Aperio Image Analysis User’s Guide
Exporting Results as Text
ToexportthetextoftheLayerAttributespanetoatextfile:OntheLayer
Attributespane,click
.Youwillbeaskedtospecifythenameandlocationof
thetextfiletobecreated.Thistextfileisatab‐delimitedfilethatcanbeimported
intoaspreadsheetprogram.
ToexportthetextoftheLayerRegionspanetoatextfile:OntheLayerRegions
pane,click
.Youwillbeaskedtospecifythenameandlocationofthetextfile
tobecreated.Thistextfileisatab‐delimitedfilethatcanbeimportedintoa
spreadsheetprogram.
Exporting Results to a Spreadsheet
ToexportthetextoftheLayerAttributespanetoaMicrosoftExcelspreadsheet:
OntheLayerAttributespane,click
.Youwillbeaskedtospecifythename
andlocationofthespreadsheet.xlsfiletobecreated.
ToexportthetextoftheRegionsAttributespanetoaMicrosoftExcel
spreadsheet:OntheRegionsAttributespane,click
.Youwillbeaskedto
specifythenameandlocationofthespreadsheet.xlsfiletobecreated.
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A
Troubleshooting
This appendix provides tips on identifying problems with
glass slides that may contribute to unsatisfactory analysis
results.
In“IntendedUse”onpage2,wediscussedtheimportanceofunderstanding
algorithmparameterssothatyoucanfine‐tunethemsotheanalysisprovidesthe
resultsyouexpectandwant.Evenafteradjustingalgorithmparameters,a
numberoffactorscanarisethatmaycausethealgorithmtoyieldunsatisfactory
resultsforyourapplication.Detectionoftheseconditionsmustbedonebya
trainedexpertuserwhocanidentifythesesituationsandtaketheappropriate
correctiveaction.Insomecases,theappropriateactionmaybetomarktheglass
slideasdefective.Inothercases,itmaybethatnewtuningofthealgorithminput
parametersisrequiredorthestaining/preparationprocessmustberepeated.
Thisappendixcontainsapartiallistofconditionsthatmayresultin
unsatisfactoryanalysisresults.
Problem Cause Effect Corrective Action
Defective
slide
Broken slide Blurry image and/or
artifacts at crack.
You should reject the
slide.
Air pockets under
coverslip.
Image very blurry, objects
too light and not easily
identifiable. The algorithm
will likely miss information
in this area.
You can exclude these
areas from analysis by
using the ImageScope
negative-pen
annotation tools
Pen marks Areas will be dark and will
obscure underlying
information.
You can exclude these
areas from analysis by
using the ImageScope
negative-pen
annotation tool.
Wrong
tissue type
Algorithm tuned
for one type of
tissue (for
example, breast
cancer) and
operator runs it
on another type
of tissue (for
example, liver).
The algorithm analysis will
still give results, but the
results will likely be
meaningless.
Choose a different
algorithm or input
parameter set.
Aperio Image Analysis User’s Guide 39
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Appendix A - Troubleshooting
40 Aperio Image Analysis User’s Guide
Problem Cause Effect Corrective Action
Wrong
regions
designated
for analysis
For nuclear
analysis, stromal
and lymphocytes
were included in
the designated
regions.
Nuclear counts will be too
large.
Operator must
recognize these areas
and annotate them to
exclude them from
analysis.
Bad
histology
preparation
Tissue sections
too thick.
Image will be blurry all
over.
Prepare new slide.
Folds in tissue. Blurry areas and cells that
are superimposed on each
other.
Prepare new slide or
annotate these areas to
exclude them from
analysis.
Variations
in staining
process
Staining is too
light.
Cells are not solidly
stained and the algorithm
may over count by
subdividing single cells
into multiple cells.
Adjust input
parameters if possible
or prepare a new slide.
Staining is too
dark.
Cells which are close
together become merged
and counted as a single
cell.
Adjust input
parameters if possible
or prepare a new slide.
Staining is not
specific to the
features being
analyzed.
For nuclear analysis,
significant cytoplasmic
staining may cause cells to
be misidentified.
Prepare new slide.
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Index
algorithminstallation,7
algorithmmacro,21
algorithmtuningwindow,28
analysis,31
creatingnewmacro,22
savingmacro,26
testing/modifyingmacros,27
annotations
exporting,38
measuring,19
viewing,19
Annotationswindow
detailedview,3
summaryview,3
Aperioreleaserequirements,2
batchanalysis,33
Colocalizationalgorithm,2
ColorDeconvolutionalgorithm,2
creatingnewalgorithmmacro,22
digitalslide,1
analyzing,31
openinginSpectrum,10
openinglocal,12
openingremote,14
exportingresults,38
asspreadsheet,38
astext,38
FDAclearance,2
IHCanalysis,3
ImageScope,1
advancedfeatures,9
drawingtools,18
installation,7
intendeduse,3
introduction,1
IQ,9
macro,21
creatingnew,22
saving,26
monitorrequirements,2
openingdigitalslides,12,14
openingremotedigitalslide,14
PositivePixelCountalgorithm,1
prerequisites,2
RareEventDetectionalgorithm,2
registeringmacrosonSpectrum,26
runningthealgorithm,31
selectingareastoanalyze,18
slide‐specificprocessing,3
troubleshooting,39
Aperio Image Analysis User’s Guide 41
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Aperio Image Analysis User’s Guide
MAN-0013, Revision C
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