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Clinical Specular Microscopy Corneal Endothelial Cell Morphology
Bernard E. McCarey, Ph.D.
Emory University Eye Center
Atlanta, Georgia U.S.A.
FDA 2001
Clinical Specular Microscopes Available
Contact AnalysisKeller-Konan: SP-580 manual photo digitizedHAI Labs: HAI CL-1000xyz manual or automaticTOMEY: EM-1000 automatic
Non-ContactBio Optics: LMS-12000 automatic with manualTopcon: SP-2000P automatic limitedKonan: ROBO Pachy SP-9000 manual digitalKonan: ROBO CA SP-8800 manual digital
Clinical Specular Microscopes Available
Contact Non-Contact
instrument Keeler-Konan Konan ROBO
topical anesthetic yes no
focusing subjective: visual automatic
alignment difficulties focus & applanation corneal curvature
field size 0.32 to 0.135 mm2 0.08 mm2
(0.9 x 0.35) to (0.9 x 0.15 mm) (0.35 x 0.23 mm)
no. cells per field at 2500 cells/mm2 700 to 800 cells 160 cells
Corneal Endothelial Cell Morphology
• Cell Area ± S.D. (µm2)
• Cell Density (cells / mm2)
• Polymegethism (CV)
• Pleomorphism (% 6 sided)
Corneal Endothelial Cell Morphology
cells
Cell Density Coefficient of Variation
=1 x 106
µm2
mm2
Mean cell area, µm2CV =
Mean cell area, µm2
SDcell area
mm2
Corneal Endothelial Cell Density
• 350,000 cell / cornea
• at birth, 3000 - 4000 cell / mm2
• at middle age, 2500 cell / mm2
• at old age, 2000 cells / mm2
• minimal acceptable, 1500 cell / mm2
• potential corneal edema, 800 cells / mm2
Yee etal; Curr. Eye Res. 4(6):6712-678, 1985
Corneal Endothelial Cell Polymegethism, CV
• Normal young adult, 0.27 to 0.28
• Literature convention uses 27 to 28
Endothelial Cell Morphology Changes Caused by Surgical Trauma
• Do localized changes effect other zones on the cornea?
• What is the chronological healing response?
• Can central Cell Density adequately document the trauma?
Individual Healing Following Intraocular Surgery in the Human
With phaco. (n=34) and extracap.
(n=25), there was an acute CD decrease at incision site with delayed evidence of
extensive cell migration. .
control 2-6 7-12 12-24 24-36 36-48 48-60 60-102
Post Operative Time (months)
0
1000
2000
3000
En
doth
elia
l Cell
Den
sity
Hoffer,KJ: Vertical Endothelial Cell DisparityAmer J Ophthalmol 87:344-349, 1979
superior
central
inferior
n=28 n=2n=23 n=4 n=7 n=7 n=6 n=3
Hoffer 1970
Serial Healing Following ICCE Matsuda, Suda and Manabe; Amer J Ophthalmol 98:313-319, 1984
Serial Healing Following Keratoplasty Matsuda, Suda and Manabe; Amer J Ophthalmol 98:313-319, 1984
Endothelial Cell Changes Caused by Contact Lenses
• Transient (bleb) Morphology Changes• Chronic Morphology Changes
– Pleomorphism (%H)– Polymegethism (CV)
Schoessler; J Am Opto Assoc. 58(10):804-810, 1987
Polymegethism Endothelial Morphology
CV=45 CD=3268 CV=76 CD=2967 CV=58 CD=3121
Corneal Endothelial Cell Density Determination
• Comparison Method: compare to known “honey comb” pattern• Frame Method: count the number of cell within a frame• Corner Method: determine cell area from a polygon digitization by
locating cell border intersections• Center Method: determine cell area from adjacent polygon centers,
“center to center”
Frame Method
• Count all cells within a frame
• Adjust for cells extending outside of frame
• Count partial cells as full cells on 2 adjacent frame sides
• Convert cells counted per partial mm2 to cells / mm2
Frame Method Accuracy
• Size of Frame: determines number of cells to be counted
• Decision on Partial Cells• Decision on Cell Borders• Blue Frame frame = 0.036 mm2
• Yellow Frame frame = 0.018 mm2
2502 cellsmm2 = 90
cellsframe X 27.8*
2502 cellsmm2 = 45
cellsframe X 55.6*
* multiplier = 1/ frame, mm2
Center Method *
• Dot center of contiguous cells
• Ideally count in a circle
• In practice count in a rectangle.
* Konan Software
Center Method: off centered dots
Analysis of approx. 100 cells, n= 5Off centered 1 to 10 cells per frame
Average Error ranged -0.2% to +0.1%
*
**
***
***
**
% change =CD decenter - CDcenter
CDcenter
X 100
Center Method: omitted cells
Endothelial Cell Image Perfect Hexagon Pattern
cell size % change big -1.1 medium -2.0 small -2.9
cell size % change small -0.9 small -1.9 small -2.7
Cell omitted % change -1 -1.1 -2 -2.1 -3 -3.2 -4 -4.4
% change = CDomit - CDfull
CDfull
X 100
Frame and Center Methods*
variable morphology vs number of cells analyzed
CV = 25
* Konan software
Frame and Center Methods*
Cell Density (CV=25) vs number of cells analyzed
* Konan software
0 25 50 75 100 125 150 175 200
Number of Cells in Final Calculation
2000
2500
3000
3500
4000
Cell
Den
sity
(ce
lls/m
m^
2)
Y=3090 +/- 50
X=150
n=16
Frame and Center Methods*
Coef. of Variation (CV=25) vs number of cells analyzed
* Konan software
0 25 50 75 100 125 150 175 200
Number of Cells in Final Calculation
0
10
20
30
40
50
60
Coeffi
cien
t of
Vari
ati
on
(C
V)
Y=25 +/- 2
X=150
n=16
Frame and Center Methods*
variable morphology vs number of cells analyzed
CV = 45
* Konan software
Frame and Center Methods*
Cell Density (CV=45) vs number of cells analyzed
* Konan software
0 25 50 75 100 125 150 175 200
Number of Cells in Final Calculation
1500
2000
2500
3000
3500
Cell
Den
sity
(ce
lls/m
m^
2)
Y=2607 +/- 162
X=150
n=16
S.D. is 3 x larger than S.D. of CV 25 endothelium
Frame and Center Methods*
Coef. of Variation (CV=45) vs number of cells analyzed
* Konan software
0 25 50 75 100 125 150 175 200
Number of Cells in Final Calculation
0
10
20
30
40
50
60
Coeffi
cien
t of
Vari
ati
on
(C
V)
Y=45 +/- 3
X=150
n=16
Effect of Number of Cells per Image verses Coefficient of Variation
• S.D. increases with increasing CV• S.D. decreases with increasing number of cells counted• S.D. stabilizes with >100 cells counted • S.D. reflects an always present data spread• Konan software max. count of 200 cells
Cell Area ± S.D.
Center Method * Calculation
* Konan Software
7 Dotted1 Final0 25 50 75 100 125 150 175 200
Number of Dotted Cells
0
30
60
90
120
150
Nu
mb
er
of C
ells
in F
ina
l Co
un
t
Y = +0.795 X -13.8
Max. Number Countable in Cell Density Field Konan Non-Contact Specular Microscope
Good vs Poor Quality Images
Dot Maximum Number of
Cells per Field
Final Number Less Than
Number Dotted
0 500 1000 1500 2000 2500 3000 3500 4000
Cell Density (cells per sq. mm)
0
50
100
150
200N
um
be
r o
f Ce
lls in
Fin
al C
ou
nt
Y = +0.0462X -3.4
n = 123
Poor
Images
Good Images
Image Location with Konan Specular Microscope
CD=2437 CV=31
AB
C
= centralA = 0.5mm (1mm dia.)B = 1mm (2mm dia.)C = 2mm (4mm dia.)
Center image
A
B
C
Image Location within a 4 mm Diameter Zone
CD=2532 CV=31
AB
C
Image at 4mm
image locationn = 13One Way Anal. of Var.p = 0.000378Est. of Variation = 13.1%
*
*** *
***
*
*
*
**
*
*
EOV = CDmax - CDmin
CDmaxX 100
Image Location within a 3 mm Diameter Zone
CD=2531 CV=31
AB
C
*** *
*****
**
* image locationn = 13One Way Anal. of Var.p = 0.00118Est. of Variation = 14.9%
* Image at 3mm
*
Image Location within a 2 mm Diameter Zone
CD=2522 CV=34
AB
C*
** ****
**
**
* image locationn = 13One Way Anal. of Var.p = 0.241Est. of Variation = 9.4%
* Image at 2mm
*
Image Location within a 1 mm Diameter Zone
CD=2315 CV=32
AB
C***
* ***
**
image locationn = 9One Way Anal. of Var.p = 0.284Est. of Variation = 8.1%
* Image at 1mm
Image Location at Center
CD=2479 CV=31
AB
C
Center imagecenter image locationn = 10One Way Anal. of Var.p = 0.981Est. of Variation = 4.2%
Control Endothelial Cell Density Repeatability Range
• Control eyes from Medennium Corp. Clinical Trial
• A single Reading Center
• 58 subjects from 7 clinical sites in USA
• Subjects CV= 36 +/- 6 (mean +/- SD)
• Images captured at baseline and 3 months
• Repeatability was determined between each time period
• Paired t-test between baseline and 3 mo. was p=0.727
Control Endothelial Cell Density Repeatability Range
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+/- % Difference ((3 month - Baseline) x 100)
0
25
50
75
100
Cu
mu
lativ
e T
ota
l Su
bje
cts
(%)
Baseline
Interpretation:
Y-axis is the % of subjects with X% difference between base-line and 3 mo.
Control Endothelial Cell Density Repeatability Range
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+/- % Difference ((3 month - Baseline) x 100)
0
25
50
75
100
Cu
mu
lativ
e T
ota
l Su
bje
cts
(%)
Baseline
Interpretation:
50% within +/- 2.5%
100% within +/- 9%
Specular Microscopy Clinical Site Issues
• Clinical Trial Criteria– Coordinator Experience– Criteria for quality data
• Specular Microscope– Model and software– Experience
• Image Capture– Ophthalmic photographer, dedicated to image capturing – Ophthalmic technician, diverse patient care – Technician, limited training
• Clinical locations• Clinical Site Training
Clinical Site Training
• All sites should have the same microscope model• Automated non-contact specular microscope• Preferably one technician per site• Require and evaluate practice efforts
– central images of same individual– multiple images to establish individual statistics
• Re-evaluate practice efforts over time• Repeatability of skill is key to good data• Individual training visit promotes uniformity of skills
The End
Thanks to Michael Lynn for statistical assistance Jill Walter for technical assistance
