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 manual photo digitized
HAI Labs: HAI CL-1000xyz manual or automatic
TOMEY: EM automatic
Non-Contact
Bio Optics: LMS automatic with manual
Topcon: SP-2000P automatic limited
Konan: ROBO Pachy SP manual digital
Konan: ROBO CA SP 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 to mm 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/mm to 800 cells 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 =
mm2
Mean cell area, µm2
Mean cell area, µm2

6. Corneal Endothelial Cell Density
350,000 cell / cornea
at birth, cell / mm2
at middle age, cell / mm2
at old age, cells / mm2
minimal acceptable, cell / mm2
potential corneal edema, 800 cells / mm2
Yee etal; Curr. Eye Res. 4(6): , 1985

7. Corneal Endothelial Cell Polymegethism, CV
Normal young adult, 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. (n=34) and extracap. (n=25), 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 98:313-319, 1984

11. Serial Healing Following Keratoplasty Matsuda, Suda and Manabe; Amer J Ophthalmol 98:313-319, 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): , 1987

13. Polymegethism Endothelial Morphology
CV=45 CD=3268
CV=76 CD=2967
CV=58 CD=3121

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. * multiplier = 1/ frame, 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 = mm2
Yellow Frame frame = 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 size % change
big
medium
small
Cell omitted % change
cell size % change
small
small
small
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 (CV=25) vs number of cells analyzed
* Konan software

22. Frame and Center Methods*
Coef. of Variation (CV=25) 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 (CV=45) 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 (CV=45) 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 >100 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
CD=2437 CV=31
= 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
One Way Anal. of Var.
p =
Est. of Variation = 13.1% * * *
CD=2532 CV=31 * *
Image at 4mm B * * * * * A C * * * *
CDmax - CDmin
EOV =
X 100 *
CDmax

31. Image Location within a 3 mm Diameter Zone
One Way Anal. of Var.
p =
Est. of Variation = 14.9% *
CD=2531 CV=31 * *
Image at 3mm * * B * * * * * A * * C * *

32. Image Location within a 2 mm Diameter Zone
One Way Anal. of Var.
p = 0.241
Est. of Variation = 9.4%
CD=2522 CV=34 * *
Image at 2mm * * * B * * * * * A * * * C *

33. Image Location within a 1 mm Diameter Zone
One Way Anal. of Var.
p = 0.284
Est. of Variation = 8.1%
CD=2315 CV=32 *
Image at 1mm * * * B * * * * * * A C

34. Image Location at Center
center image location
n = 10
One Way Anal. of Var.
p = 0.981
Est. of Variation = 4.2%
CD=2479 CV=31
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 p=0.727

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