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 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

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 Cell Density Coefficient of Variation µm2 mm2 1 x 106 SDcell area cells = CV = mm2 Mean cell area, µm2 Mean cell area, µm2

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. .

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

* 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 = 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

* 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= 5 Off centered 1 to 10 cells per frame Average Error ranged -0.2% to +0.1% CD decenter - CDcenter * * * * % change = X 100 * * * * * * * CDcenter

Center Method: omitted cells Endothelial Cell Image Perfect Hexagon Pattern cell size % change big -1.1 medium -2.0 small -2.9 Cell omitted % change -1 -1.1 -2 -2.1 -3 -3.2 -4 -4.4 cell size % change small -0.9 small -1.9 small -2.7 CDomit - CDfull % change = X 100 CDfull

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

Frame and Center Methods* Coef. of Variation (CV=25) vs number of cells analyzed * Konan software

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 S.D. is 3 x larger than S.D. of CV 25 endothelium * Konan software

Frame and Center Methods* Coef. of Variation (CV=45) vs number of cells analyzed * Konan software

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

Center Method * Calculation 7 Dotted 1 Final * Konan Software

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

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

Image Location within a 4 mm Diameter Zone One Way Anal. of Var. p = 0.000378 Est. of Variation = 13.1% * * * CD=2532 CV=31 * * Image at 4mm B * * * * * A C * * * * CDmax - CDmin EOV = X 100 * CDmax

Image Location within a 3 mm Diameter Zone One Way Anal. of Var. p = 0.00118 Est. of Variation = 14.9% * CD=2531 CV=31 * * Image at 3mm * * B * * * * * A * * C * *

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 *

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

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

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 Interpretation: Y-axis is the % of subjects with X% difference between base-line and 3 mo.

Control Endothelial Cell Density Repeatability Range 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