Clinical Specular Microscopy Corneal Endothelial Cell Morphology

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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
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Clinical Specular Microscopes Available
Contact Analysis Keller-Konan SP-580
manual photo digitized HAI Labs HAI CL-1000xyz
manual or automatic TOMEY EM-1000
automatic
Non-Contact Bio Optics LMS-12000
automatic with manual Topcon SP-2000P
automatic limited Konan ROBO Pachy SP-9000
manual digital Konan ROBO CA SP-8800
manual digital
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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
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Corneal Endothelial Cell Morphology

• Cell Area S.D. (µm2)
• Cell Density (cells / mm2)
• Polymegethism (CV)
• Pleomorphism ( 6 sided)

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Corneal Endothelial Cell Morphology
Cell Density
Coefficient of Variation
µm2 mm2
1 x 106
SDcell area
cells

CV
Mean cell area, µm2
Mean cell area, µm2
mm2
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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
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Corneal Endothelial Cell Polymegethism, CV

• Normal young adult, 0.27 to 0.28
• Literature convention uses 27 to 28

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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?

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Individual Healing Following Intraocular Surgery
in the Human
With phaco. (n34) and extracap. (n25), there
was an acute CD decrease at incision site with
delayed evidence of extensive cell migration. .
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Serial Healing Following ICCE Matsuda, Suda and
Manabe Amer J Ophthalmol 98313-319, 1984
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Serial Healing Following Keratoplasty Matsuda,
Suda and Manabe Amer J Ophthalmol 98313-319,
1984
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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
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Polymegethism Endothelial Morphology
CV45 CD3268
CV76 CD2967
CV58 CD3121
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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

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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

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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

cells
cells
2502
90
X 27.8
mm2
frame
cells
cells
2502
45
X 55.6
mm2
frame
multiplier 1/ frame, mm2
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Center Method

• Dot center of contiguous cells
• Ideally count in a circle
• In practice count in a rectangle.

Konan Software
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Center Method off centered dots
Analysis of approx. 100 cells, n 5 Off centered
1 to 10 cells per frame Average Error
ranged -0.2 to 0.1
CD decenter - CDcenter




change
X 100







CDcenter
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Center Method omitted cells
Endothelial Cell Image
Perfect Hexagon Pattern
Cell omitted change -1
-1.1 -2
-2.1 -3 -3.2
-4 -4.4
cell size change big
-1.1 medium -2.0
small -2.9
cell size change small
-0.9 small -1.9
small -2.7
CDomit - CDfull
change
X 100
CDfull
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Frame and Center Methods variable morphology vs
number of cells analyzed
CV 25
Konan software
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Frame and Center Methods Cell Density (CV25) vs
number of cells analyzed
Konan software
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Frame and Center Methods Coef. of Variation
(CV25) vs number of cells analyzed
Konan software
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Frame and Center Methods variable morphology vs
number of cells analyzed
CV 45
Konan software
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Frame and Center Methods Cell Density (CV45) vs
number of cells analyzed
S.D. is 3 x larger than S.D. of CV 25 endothelium
Konan software
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Frame and Center Methods Coef. of Variation
(CV45) vs number of cells analyzed
Konan software
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Effect of Number of Cells per Image verses
Coefficient of Variation
Cell Area S.D.

• S.D. increases with increasing CV
• S.D. decreases with increasing number of cells
  counted
• S.D. stabilizes with gt100 cells counted
• S.D. reflects an always present data spread
• Konan software max. count of 200 cells

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Center Method Calculation
7 Dotted 1 Final

Konan Software
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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
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Image Location with Konan Specular Microscope
CD2437 CV31
central A 0.5mm (1mm dia.) B 1mm (2mm
dia.) C 2mm (4mm dia.)
Center image
B
C
A
A
B
C
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Image Location within a 4 mm Diameter Zone
image location n 13 One Way Anal. of Var. p
0.000378 Est. of Variation 13.1



CD2532 CV31


Image at 4mm
B





A
C




CDmax - CDmin
EOV

X 100
CDmax
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Image Location within a 3 mm Diameter Zone
CD2531 CV31

image location n 13 One Way Anal. of Var. p
0.00118 Est. of Variation 14.9


Image at 3mm


B





A

C



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Image Location within a 2 mm Diameter Zone
CD2522 CV34
image location n 13 One Way Anal. of Var. p
0.241 Est. of Variation 9.4


Image at 2mm



B





A



C

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Image Location within a 1 mm Diameter Zone
CD2315 CV32
image location n 9 One Way Anal. of Var. p
0.284 Est. of Variation 8.1

Image at 1mm



B




A


C
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Image Location at Center
CD2479 CV31
center image location n 10 One Way Anal. of
Var. p 0.981 Est. of Variation 4.2
Center image
B
A
C
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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
  p0.727

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Control Endothelial Cell Density Repeatability
Range
Interpretation Y-axis is the of subjects with
X difference between base-line and 3 mo.
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Control Endothelial Cell Density Repeatability
Range
Interpretation 50 within /- 2.5 100
within /- 9
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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

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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

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The End
Thanks to Michael Lynn for statistical
assistance
Jill Walter for technical assistance