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Clinical Specular Microscopy Corneal Endothelial Cell Morphology

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... 27 to 28 Endothelial Cell Morphology Changes Caused by Surgical Trauma Do localized changes effect other zones on the cornea? – PowerPoint PPT presentation

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Title: Clinical Specular Microscopy Corneal Endothelial Cell Morphology


1
Clinical Specular Microscopy Corneal
Endothelial Cell Morphology
  • Bernard E. McCarey, Ph.D.
  • Emory University Eye Center
  • Atlanta, Georgia U.S.A.

FDA 2001
2
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
3
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
4
Corneal Endothelial Cell Morphology
  • Cell Area S.D. (µm2)
  • Cell Density (cells / mm2)
  • Polymegethism (CV)
  • Pleomorphism ( 6 sided)

5
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
6
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
7
Corneal Endothelial Cell Polymegethism, CV
  • Normal young adult, 0.27 to 0.28
  • Literature convention uses 27 to 28

8
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?

9
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. .
10
Serial Healing Following ICCE Matsuda, Suda and
Manabe Amer J Ophthalmol 98313-319, 1984
11
Serial Healing Following Keratoplasty Matsuda,
Suda and Manabe Amer J Ophthalmol 98313-319,
1984
12
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
13
Polymegethism Endothelial Morphology
CV45 CD3268
CV76 CD2967
CV58 CD3121
14
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

15
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

16
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
17
Center Method
  • Dot center of contiguous cells
  • Ideally count in a circle
  • In practice count in a rectangle.


Konan Software
18
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
19
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
20
Frame and Center Methods variable morphology vs
number of cells analyzed
CV 25
Konan software
21
Frame and Center Methods Cell Density (CV25) vs
number of cells analyzed
Konan software
22
Frame and Center Methods Coef. of Variation
(CV25) vs number of cells analyzed
Konan software
23
Frame and Center Methods variable morphology vs
number of cells analyzed
CV 45
Konan software
24
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
25
Frame and Center Methods Coef. of Variation
(CV45) vs number of cells analyzed
Konan software
26
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

27
Center Method Calculation
7 Dotted 1 Final

Konan Software
28
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
29
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
30
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
31
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



32
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

33
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
34
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
35
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

36
Control Endothelial Cell Density Repeatability
Range
Interpretation Y-axis is the of subjects with
X difference between base-line and 3 mo.
37
Control Endothelial Cell Density Repeatability
Range
Interpretation 50 within /- 2.5 100
within /- 9
38
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

39
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

40
The End
Thanks to Michael Lynn for statistical
assistance
Jill Walter for technical assistance
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