USE OF THE KONAN NONCON-USE OF THE KONAN NONCON-ROBO SPECULAR MICROSCOPE ROBO SPECULAR MICROSCOPE
IN CLINICAL RESEARCHIN CLINICAL RESEARCH
Henry F. Edelhauser, Ph.D.Ramzy G. Azar, MPH
Emory University Eye CenterAtlanta, Georgia
PurposePurpose
Understand variability issues with Understand variability issues with specular microscopy that may bias specular microscopy that may bias resultsresults
ObjectivesObjectives
Provide examples of good and poor Provide examples of good and poor photographyphotography
Illustrate variability in specular Illustrate variability in specular microscopy photography and microscopy photography and analysisanalysis
Illustrate variability within a single Illustrate variability within a single imageimage
What is a Good Image?What is a Good Image? Distinct cellsDistinct cells Can identify at least Can identify at least
150 cells150 cells Cells can be Cells can be
grouped in a grouped in a uniform areauniform area
What may be good What may be good for clinical purposes for clinical purposes may not be good may not be good researchresearch
Things to Consider That Things to Consider That Affect Quality of ImageAffect Quality of Image
Dry eyeDry eye Contact lens useContact lens use Wrong Specular Manual SettingsWrong Specular Manual Settings KeratoconusKeratoconus Patient CompliancePatient Compliance AgeAge Training, experience of Training, experience of
photographerphotographer
Poor Quality ImagesPoor Quality Images
Poor Quality Images Poor Quality Images Continued…Continued…
Poor Quality Images Poor Quality Images Continued…Continued…
Conditions that Potentially Conditions that Potentially Increase VariabilityIncrease Variability
Guttata (Fuch’s dystrophy)Guttata (Fuch’s dystrophy) Polymegethism/PleomorphismPolymegethism/Pleomorphism InjuryInjury Low Cell density (Huge cells)Low Cell density (Huge cells)
Guttata (Fuch’s dystrophy)Guttata (Fuch’s dystrophy)
Capturing the Best Capturing the Best Image PossibleImage Possible
Make sure Pt is comfortableMake sure Pt is comfortable Instruct Pt to blinkInstruct Pt to blink Instruct Pt not to move and to open eyes Instruct Pt not to move and to open eyes
widewide Instruct Pt to focus on the green lightInstruct Pt to focus on the green light Be patientBe patient Use Manual setting to improve quality when Use Manual setting to improve quality when
cornea is unusually thicker than normalcornea is unusually thicker than normal
Things to Consider When Things to Consider When Analyzing ImagesAnalyzing Images
Locate the best and most representative Locate the best and most representative areaarea– Number of cellsNumber of cells– Quality of CellsQuality of Cells
No shadowsNo shadows DiseaseDisease
– Use area with the fewest distortionsUse area with the fewest distortions BlurringBlurring Washed-out imagesWashed-out images ShadowsShadows
Locating the Best Analysis Locating the Best Analysis Area (Sample Images)Area (Sample Images)
Dotting CellsDotting Cells
Dot all Cells at the CenterDot all Cells at the Center– Remain accurate and consistent Remain accurate and consistent
throughoutthroughout Dot 150 cellsDot 150 cells Grouping is importantGrouping is important
Where to Group the Where to Group the Analysis?Analysis?
What is Wrong With This What is Wrong With This Analysis?Analysis?
Analysis is not Analysis is not representative representative
Introducing Introducing BiasBias
Not likely to Not likely to repeatrepeat
Not enough Not enough cells countedcells counted
Grouping DetailsGrouping Details
EasyEasy ClearClear No No
shadowshadowss
Dot 150 Dot 150 cellscells
Need Need Good rep.Good rep.
Take Take more more timetime
Dot > 150Dot > 150
NormalNormal PolymegathismPolymegathism
Grouping your AnalysisGrouping your Analysis
Correct GroupingCorrect Grouping– ConcentricConcentric– EvenEven– UniformUniform
Incorrect GroupingIncorrect Grouping– linearlinear– unevenuneven– WindingWinding
Cell Grouping - GuttataCell Grouping - Guttata
Group only in one areaGroup only in one area
To Analyze the Cells:To Analyze the Cells: You need to be able to visualize You need to be able to visualize
cellscells Find a patternFind a pattern IdentifyingIdentifying
– Cells vsCells vs– Damage vsDamage vs– ShadowsShadows
Where an Image is Where an Image is Analyzed Can Create Analyzed Can Create
VariabilityVariability
Examples of VariabilityExamples of Variability
CD = 2873CD = 2873
SD = 170SD = 170
CV = 48CV = 48
6A = 536A = 53
CD = 2976CD = 2976
SD = 113SD = 113
CV = 33CV = 33
6A = 536A = 53
ΔCD = 103
(4%)
Examples of Variability Examples of Variability Within Readers Within Readers
CD = 2531CD = 2531
SD = 139SD = 139
CV = 35CV = 35
6A = 556A = 55
CD = 2358CD = 2358
SD = 222SD = 222
CV = 52CV = 52
6A = 566A = 56
ΔCD = 173
(7%)
Examples of Variability Examples of Variability Between ReadersBetween Readers
AnalysisAnalysisRepeated Repeated
4x4x#1 - 2631#1 - 2631
#2 - 2557#2 - 2557
#3 - 2531#3 - 2531
#4 - 2570#4 - 2570
#5 - 2624#5 - 2624
Range 2531 - 2631Range 2531 - 2631
Consequences of Under or Consequences of Under or Over CountingOver Counting
Analysis N p -valueMean SD (Cells) %
Regular 10 3233 24SKIP
One 8 3213 49 0.3174 -20 -0.6Two* 8 3188 20 0.0006 -45 -1.4Three 4 3137 9 <0.0001 -96 -3.0Four 4 3091 30 <0.0001 -142 -4.4Five 4 3069 25 <0.0001 -164 -5.1
COUNTED TWICE
One 10 3240 30 0.5741 7 0.2Two 7 3255 19 0.0613 22 0.7
Three* 6 3257 13 0.0429 24 0.7Four 6 3265 35 0.0457 32 1.0Five 6 3281 36 0.0062 48 1.5
Study by Nauman Rashid
CD (Cells/mm2) CD
Endothelial Cell DensityEndothelial Cell Density
0
1000
2000
3000
4000
5000
10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89
Age (years)
Cel
l Den
sity
(cel
ls/m
m2 )
non-contact ROBO
contact (Yee et al.)
Asian population
pre-LASIK (Jones et al.)
Japanese population (Matsuda et al.)
Precision of 36 Robo corneal endothelial Precision of 36 Robo corneal endothelial specular images of each eye (OD, OS) specular images of each eye (OD, OS)
taken on 18 different days and analyzed taken on 18 different days and analyzed with the Robo softwarewith the Robo software
NN Cell DensityCell Density PrecisioPrecisionn
ODOD 1818 2545 ± 45 2545 ± 45 cells/mmcells/mm22 (1.7%)(1.7%)
OSOS 1818 2600 ± 41 2600 ± 41 cells/mmcells/mm22 (1.5%)(1.5%)
(From AJO 125:465-471, 1998 LASIK Paper)
Age Dependent Cell Density Age Dependent Cell Density Variation Within 3 Different Variation Within 3 Different
Corneal RegionsCorneal Regions
y = -18x + 3454
y = -14x + 3622
y = -13x + 4310
1000
2000
3000
4000
5000
6000
10 20 30 40 50 60 70 80
Age (Years)
Cel
l Den
sity
(cel
ls/m
m2)
Central
Paracentral
Limbal
(10)(11)
(9)
(9)
(14)
(11) (9)
Sources of Variability Sources of Variability SummarySummary
Difficult to return to same location Difficult to return to same location (1 mm = (1 mm = 56 cells/mm 56 cells/mm2 2 - - 2.0%2.0%))
Poor image quality (minimal # of Poor image quality (minimal # of analyzable cells = analyzable cells = 100100) )
Technician error Technician error (Training/consistency)(Training/consistency)
Reader analysis (Training/consistency) Reader analysis (Training/consistency) Equipment calibration/alignmentEquipment calibration/alignment
Data Flow ChartData Flow Chart
Reading Reading CenterCenter Data Process Data Process
CenterCenter
Specular Specular SitesSites
Technology CoTechnology Co
