…Restoring Sight Worldwide

2012 R. TOWNLEY PATON LECTURE

CORNEA PRESERVATION AT 4⁰C:

AN AMERICAN STORY

Presented by Jonathan H. Lass, MD, during the

EBAA/Cornea Society Fall Educational

Symposium

Friday, November 9, 2012

Palmer House Hilton – Chicago, Illinois

1

Jonathan H. Lass, M.D.

Charles I Thomas Professor and Chairman

CWRU Department of Ophthalmology and Visual Sciences

Director, University Hospitals Eye Institute

Medical Director, Cleveland Eye Bank

Medical Director, Cornea Image Analysis Reading Center

R. Townley Paton Award Lecture 2012

Cornea Preservation at 4°C: An American Story

Financial Disclosures

� No disclosures related to this presentation

� Cornea Image Analysis Reading Center at University Hospitals

Eye Institute and Case Western Reserve University receives

grant support from the following companies:

– Eyetech

– Glaukos

– Ivantis

– Regeneron

– Transcend Medical

and NEI grant support: U10EY012358 and U10EY020798

2

Acknowledgements

Howard LeibowitzClaes Dohlman

Debbie Pavan Langston Richard ThoftKen Kenyon

Hank EdelhauserBill Bourne

Edward PurnellBill Reinhart

Beth Ann BenetzLoretta Szczotka Roy Beck

Maryann RedfordAllison Edwards Steve Oversby

Paton Award RecipientsClaes Dohlman (1986)

B Ahmad (1987)

D Doughman (1987)

P Laibson

W Reinhart (1998)

J Lass (2012)

M Hettinger (2006)

W Chu (2004)

R. Lindstrom (1995) T Lindquist (2009)

J Krachmer (1991)

P Dubord (2001)

M Macsai (2003)

M Mannis (1996)

E Holland (2002)

D Cavanaugh (2000)

W Van Meter (2010)

Herbert Kaufmann (1983)

F Brightbill (1988)

W Bourne (1994)

J Sugar (1997)

A Sugar (2011)

2/3rds of the 31 Paton award recipients,since establishment of the award in 1982,can trace their lineage to the cornea fellow-ships of Paton awardees, Dr. Herbert Kauf-man (1983) and Dr. Claes Dohlman (1986). Their scientific contributions to the field were matched by their immense contribu-tions to training the next generation of leaders in eye banking and corneal trans-plantation.

3

Acknowledgements

Other Collaborators on Preservation Media

and Keratoplasty Studies� KSol vs CSM: Bruner, Sanders

� CSM vs DexSol: Bruner, Lindstrom, Skelnik

� Optisol vs DexSol: Bruner, Cano, Laing, Maguire, Meyer, Musch, Soong, A. Sugar

� Optisol GS: Gordon, Meyer, Norden, Soong, Sugar

� ProCell vs Optisol: Gordon, Laing, Musch and the Cornea Preservation Study Group

(CWRU, Price Vision, OSU, Iowa, Louisville, Michigan, Wayne State and supporting

eye banks

� Cornea Donor Study and Specular Microscopy Ancillary Study: Beck, Gal, Holland,

Mannis, and the 41 clinical sites and 20 eye banks

� DSEK vs PKP studies: S. Debanne, M. Gorovoy, F. Price, M. Price and SMAS and

Indiana and Tampa eye banks

� Life 4˚C vs. Optisol GS: S. Debanne, F. Price, M. Price, G. Rosenwasser, M.

Rosenwasser, D. Verdier and Midwest, Indiana, and Gift of Life Eye Banks

� Cornea Preservation Time Study: Beck, Edwards and the CPTS Study Group (36

clinical sites and 23 eye banks)

Cornea Preservation at 4°C: An American Story

4

R. Townley Paton, M.D. and the

First Eye Bank in the World: A Game Changer

The Eye Bank for Sight Restoration

• Written consent for enucleation

• Harvest, process, and distribute

highest quality corneas

• Medical policies by medical

personnel

Growth of Eye Banking

Federalism vs State/Local Control

• Federalism

• Strong central governments

• Centralized eye banking with

development of organ culture

• Decentralized control of many governmental functions

with continuing debate on level of federalism

• Decentralized eye banking with initially local and

subsequent regional and national cooperation

fostered by 4˚C preservation developments

5

Central Eye Bank/Cleveland Eye Bank

� Established in 1958 at urging of

Eye Bank for Sight Restoration

under leadership of Dr. Charles I

Thomas

� Supply center working with the

Eye Bank for Sight Restoration for

16 states between Alleghenies and

Rockies

� In 1964 became Cleveland Eye

Bank

� In 1974 Dr. William Reinhart,

future Paton Award recipient,

became Medical Director

Charles I Thomas William J Reinhart

Importance of the corneal endothelium

to stromal hydration� The Giant Contributors to our

understanding in the 50’s and 60’s

� The temperature reversal phenomenon

– Davson

– Harris

– Nordquist

� Other findings on endo function

– Hedbys

– Dohlman

– Maurice

– Mishima

6

Possible Donor, Recipient, Operative, and Postoperative Factors

Influencing Endothelial Survival following Keratoplasty at 4˚C

RecipientPostoperative

FECD PBK

Operative

PKP DSEK

Graft rejection

Donor

Donor/Preoperative and Operative Factors

Influencing Endothelial Survival

after Keratoplasty at 4˚C

Donor/Preoperative

� Method of storage

� Medium

� Death to enucleation and preservation

� Preservation time

� Donor age

� Donor cell density

� Cause of death

� Recipient age

� Recipient diagnosis

– Keratoconus

– Fuchs’ dystrophy

– Pseudophakic bullous keratopathy

– HSV keratitis

– Regraft

– Chemical burn

� Glaucoma

� Synechiae

Operative

� Procedure: PKP, EK (DSEK, DMEK)

� Lens status: aphakic, AC IOL, PC IOL

� Tube shunt

7

Whole Globe storage at 4˚C

� Employed mainly until the

mid 70’s: Over 30 years

� Necessary to obtain eyes

within 6 hours of death and

place in humidified

environment at refrigerator

temperature

� Transplant within 24 to 48

hours

� Anterior trephination

necessary

Ramon Castroviejo MDPractical Implementation of 4˚C Stored

Tissue from an eye bank

8

Interview, graft, and endothelial cells 47 yrs after

receiving cornea from Paton’s eye bank and

Castroviejo’s PKP

JS with square graft and endo image 2012

Cell Loss with

Moist Chamber Storage

� 33% cell loss after one year

Keep This Cell Loss in mind: Will be

common theme

Culbertson W. Cornea 1993

9

Next Game Changer: Isolate the

cornea in a nutritious solution

similar to aqueous humor“The astonishing thing was that we had been doingtransplants for all those years with refrigerated tissuewithout realizing that the critical tissue was the endothelium.I did transplants at 2 o’clock in the morning with orthopedicnurses , and patients sat by the phone waiting to be calledwhen tissue was available. Endothelium was being bathedby an aqueous without any glucose and with dying tissue allaround and any rise in temperature during shipping wouldkill it immediately. The real insight was the idea that you hadto take off the cornea and put the endothelium in anutritious growth medium.”

Herbert Kaufman 2012

Game Changer in 4˚C Storage

McCarey-Kaufman Medium

� Developed by McCarey and

Kaufman in 1974

� TC 199 tissue culture

medium and 5% dextran to

keep cornea relatively thin

during cold storage

� Subsequently modified with

HEPES buffer and

gentamicin 100µg/ml

� With corneal dissection with

scleral rim, isolated

endothelium with nutrients to

prolong storage up to 3-4

days

Herbert Kaufman

Bernard McCarey

10

Game Changer in 4˚C Storage

McCarey-Kaufman Medium

� Still being used today in many parts of the world

� Inexpensive

� Low tech

� Suitable for use in the eye bank or in the field

� Promoted growth of local eye banks and regional

growth

� Disadvantages: No bicarb buffer, low Ca, decreased

endo and epi cell viability with increased storage time

Implementation of Tissue

Processing with MK Medium

� Took time for the eye banks

to adapt to this new approach

of storage with cornea with

scleral rim excisions in the

lab or in the field

� In 1977, three years after MK

available, I remember

preparing our donors in this

manner in the fellows room

on the Cornea Service at

MEEI

� MUSTARD PLEASE!

Fellows Room Cornea Service MEEI 1977

11

EBAA Medical Standards for Facilities

� C3.000 Facilities

– C3.100 Eye Bank Laboratory

– C3.200 Equipment, Maintenance and

Cleaning

– C3.300 Instruments and Reagents

– C3.400 Procedures Manual

– C3.500 Other Establishments Performing

Eye Banking Functions

– C3.600 Infection Control and Personnel

Safety

– C3.700 Biohazardous Waste Disposal

� E1.000 Recovery, Open-container

Processing, and Preservation

� F1.000 Tissue Evaluation

Organ culture in the United States

� Established by Donald Doughman

at Minnesota Eye Bank based on

European work

� Organ culture development

occurring concurrently as MK

medium at 4°C storage

� Organ culture storage at 37º C up

to 35 days

� Not widely implemented

– Cost with more complex

technology

– Infection concerns

� Local control over federalism won

out in the United States

Donald Doughman

Vincent Borderie

12

Specular microscopy

� Invented by Maurice

� Clinically advanced by

Bourne both in the eye

bank and postoperatively

� Led to his classic studies

on the cell loss for PKP

over 20 years

� Provided opportunity to

assess performance of

media and other donor

and recipient factors on

the donor endothelium

David Maurice William Bourne

Maurice’s original SM

Commercial development of specular

microscopy and the EBAA position

� Early developers: Laing, Koester

� Current manufacturers

– Eye banks

� HAI Tech

� Konan

– Office

� HAI Tech

� Heidelberg (confocal)

� Konan

� Nidek (confocal)

� Tomey

� Topcon

� EBAA F1.300 Medical

Standards

Specular microscopy adopted

as medical standard in 2001

over 25 years after initial

work by Bourne, Glasser and

others. Not yet a certified

procedure for the CEBT.

Vivian Xue

Sue Oak

David Glasser

13

Goals of 4°C Preservation Media

Development in the 80’s and early 90’s

� Extend preservation time beyond 5 days at 4°C

� Improve both endothelial and epithelial viability

� Reduce corneal swelling at refrigerator temperature

� Perfect to be more similar to aqueous humor

� Maintain ATP levels

� Prevent loss of stromal GAGs

� ?Induce endothelial mitotic activity and wound healing

with growth factors

Chondroitin sulfate:

The next game changer� Extends the life of the endothelium

by inhibiting the peroxidation of

the cell membrane lipids

� First described by Mizukawa and

Manabe in 1968

� Used first in organ culture then

applied to 4˚C storage

� Patented by Kaufman in 1984 with

the introduction of K-Sol

� Followed by Lindstrom and

Skelnik two years later with

development of CSM, then DexSol

Richard Lindstrom Herbert KaufmanDebbie Skelnik

14

Endothelial viability and drop out studies

Key testing approach

HCE at 4°C after 10 days HCE at 4°C after 10 days

Trypan blue (endothelial cell viability) and alizarin red S (endothelial drop out) stained

Lindstrom and Skelnik 1988

CSM K-Sol

15

4°C Preservation Media Developments

in the 80’s and early 90’sMedium K-Sol CSM DexSol ProCell

Base medium TC 199 MEM MEM MEM

CS (%) 2.5 1.35 1.35 1.35

HEPES buffer

and

bicarbonate

Yes Yes Yes Yes

Dextran 40

(%)

0 0 1 1

Na pyruvate

(mmol/L)

0 1 1 1

Additional

antioxidants

no yes yes yes

Nonessential

amino acids

no yes yes yes

Gentamicin yes yes yes yes

Insulin

(µg/ml)

no no no 10

hEGF (ng/ml) no no no 10

Clinical vision research: Epidemiologic and Biostatistical

Approaches, National Eye Institute

Personal Game Changer for Clinical Trials and

Reading Center Development

� One week course pre-ARVO in

1988

� Provided structure and methods for

well designed trials to answer

important clinical questions in

ophthalmology

� Led to design and implementation

of series of clinical trials with

paired design for examining graft

survival and cell loss with each of

the preservation media

� Development of reading center for

endothelial image analysis

Carl Kupfer

Fred Ederer Bob Sperduto

Rick Ferris

16

Our Trial Design

Corneal preservation media

� One cornea in one medium, the

other cornea in the other medium

� Recipients with same diagnosis—

all comers (FECD, PBK,

keratoconus, failed graft, scar,

dystrophy), but matched

� Comparable procedure—

phakic/phakic, AC IOL/AC IOL,

PC IOL/PC IOL, triple/triple

� Surgeries performed concurrently

� Established reading center at

CWRU/UH to determine ECD and

morphometric parameters in highly

reproducible manner

� KSol vs CSM

Ophthalmology 1989

� CSM vs. DexSol

Ophthalmology 1990

� Optisol vs. DexSol

Arch Ophthalmol 1992

� Optisol GS

AJO 1993

� ProCell vs DexSol

Ophthalmology 1994

Our Initial Prospective Trials:

KSol, CSM, DexSol

� No significant difference in paired studies for

graft success at time points out to one year

% Cell loss 4 – 6 months 12 months

KSol 9 ± 25 27 ± 22

CSM 7 ± 25 17 ± 26

DexSol 10 ±18 16 ± 23

Note: Preservation time ranged from 8-112 hours

17

ProCell: The Great Hope

� Developed by Skelnik and

Lindstrom

� Addition of insulin and EGF

to Dexsol and load donor

cornea at 4˚C prior to surgery

– Insulin to enhance cellular

metabolism and act as growth

factor for both epi and endo

– EGF as growth factor for both

epi and endo

� Cell culture data showed

mitogenic effect and ex vivo

data indicated safety out 14

days

Show mitosis slide here

HCE cells incubated with 10 ng/ml of EGF for 3 days at 35.5°CNote numerous mitotic figures

ProCell: The Great Hope

Not a game changer� Pilot study by Lindstrom of

42 PKPs with DexSol/bovine

insulin/hEGF showed

significant 13% increase in

ECD at 3 months, but

insignificant at 10% at 12

months but varying followup

and not masked.

Multicenter study comparing

ProCell to DexSol with 63 pairs

at 12 months showed:

� Comparable graft success:

95 vs. 97%

� Comparable cell loss:

12 ± 23% ProCell

16 ± 23% DexSol

Maximum storage time was 135

hours

18

Next Generation of 4°C Preservation Media

Medium DexSol Optisol Optisol GS

Base Medium MEM Hybrid of MEM

and TC-199

Hybrid of MEM

and TC-199

CS (%) 1.35 2.5 2.5

HEPES buffer and

bicarbonate

yes yes yes

Dextran 40 (%) 1 1 1

Pyruvate and

nonessential AA

yes yes yes

Ascorbic acid,

Vitamin B12,

adenosine, inosine

no yes yes

gentamicin yes yes yes

streptomycin no no yes

Bill LinkChiron Vision

Optisol and Optisol GS� Potential benefits of Optisol over

DexSol

– Endothelial viability out to 14

days

– Thinner tissue at surgery

– Better epithelial preservation

– Better protection for gram

positive organisms with

addition of streptomycin

(previously used in MK

medium)

� Safety and viability studies with

culture and ex vivo data

� Optisol introduced in 1990

� Optisol GS with

streptomycin added

introduced in 1993

� Multi-center clinical trial

comparing Optisol vs DexSol

with 31 pairs

– Optisol corneas thinner by

over 0.1 mm at surgery

– Graft success comparable at

100% vs. 97% at one year

– Cell loss comparable at one

year: 15 ± 25% Optisol

21 ± 23% DexSol

Maximum storage time was

6 days

19

Latest Development in 4°C Preservation Medium,

Life 4°C, and its storage container (Transend)Medium Life 4°C Optisol GS

Base Medium Hybrid of MEM and

TC-199

Hybrid of MEM and

TC-199

CS (%) 2.5 2.5

HEPES buffer and

bicarbonate

yes yes

Dextran 40 (%) 1 1

Pyruvate and

nonessential AA

yes yes

Ascorbic acid,

Vitamin B12,

adenosine, inosine

yes yes

Gentamicin and

streptomycin

yes yes

Glutathione yes no

Additional

Supplements B,

Stabilized L-

Glutamine,

Additional

Antioxidants,

Membrane

Stabilizers,

Micronutrients,

Trace Elements,

Vitamins,

Coenzymes

yes no

� Developed by Skelnik

� Similar to Optisol GS,

approved by the FDA for 14

days for preservation time

� Cell culture and ex vivo data

show possible improved

endothelial and epithelial

viability over Optisol GS

Optisol GS Life 4°C

Donor/Preoperative and Operative Factors Influencing Endothelial Survival after Keratoplasty at 4˚C

Donor/Preoperative

� Method of storage

� Medium

� Death to enucleation and preservation

� Preservation time

� Donor age

� Donor cell density

� Cause of death

� Recipient age

� Recipient diagnosis

– Keratoconus

– Fuchs’ dystrophy

– Pseudophakic bullous keratopathy

– HSV keratitis

– Regraft

– Chemical burn

� Glaucoma

� Synechiae

Operative

� Procedure: PKP, EK (DSEK, DMEK)

� Lens status: aphakic, AC IOL, PC IOL

� Tube shunt

20

Cornea Preservation Time Study

CPTS Clinical Sites

Wills

Jules Stein

Mid-Atlantic

Cornea

ConsultantsEye Consultants of

MD

MN Eye

Consultants

Albany

Cent Pa Eye Ctr

Eye Assoc.

Northwest

Doheny

Eye Consultants

of Atlanta

Bascom Palmer

Focal Point

VRCC &

CIARCMedical

Eye Ctr

MI Cornea

Consultants

NY Eye & Ear

NE Ohio

Eye

OSU

Northshore

Johns

Hopkins

University of IA

Hospitals

UM/Kellogg

Univ of Illinois

Verdier

Cornea Assoc.

of Texas

Dean

Eye Care of

San Diego

U of

Kentucky

Jaeb Center &

DMAC

UHCMC

Cincinnati Eye

Institute

Mayo

St. Johns’s Clinic

Univ of

Calif, San

Francisco

Moran Eye

CenterHannush

21

CPTS Eye Bank Sites

VRCC &

CIARC

Jaeb Center &

DMAC

Florida Lions

TBI

Lions Vision

Gift

Gift of Life

UT Transplant &

Lions Sight &

Tissue

Sight Society

of NE NY

Lions Eye

Bank of

Wisconsin

North Carolina Eye

Bank

Sightlife

Utah Lions Eye

Bank

Georgia Eye

Bank

Michigan Eye

Bank (Midwest) Cleveland Eye

Bank

Minnesota Lions

Eye Bank

Eye Bank for

Sight Restoration

Iowa Lions Eye

Bank

Indiana Lions Eye

Bank

Heartland Eye

Bank

Illinois Eye

Bank (Midwest)

San Diego Eye

Bank

Lions Eye Bank of

Delaware Valley

Cornea Preservation Time Study

Objectives

� To determine if the 3-year graft failure rate following DSEK performed with donor corneas with a preservation time of 8 to14 days is non-inferior to the failure rate when donor corneas with a preservation time of 7 or fewer days are used.

� To determine if the central corneal endothelial cell density 3 years after DSEK is related to preservation time.

� To evaluate donor, operative and postoperative factors on graft failure and endothelial cell density three years following DSEK.

22

CPTS treatment groups, sample size,

recruitment period and follow up� Eyes randomized to two

groups

– With a preservation time of 8 to 14 days

– With a preservation time of 7 or less days

� Optisol GS and Life 4°C are media employed.

� All clinical (surgeons, clinical staff, patients) and coordinating center masked as to preservation time and other donor factors, except the medium and the postcutthickness

� Jaeb staff is not masked, coordinating the eye banks, and is gathering the data and doing the analyses

� Sample size: 1330 eyes

� Recruitment began in April 2012

� 435 eyes randomized into the study to date

� Follow up: Three years

� Expect to report results in 2017

Cornea Preservation Time

Advantages of extending beyond 7-8 days

� Improve efficiency of tissue evaluation and distribution and reduce need for export

� Change attitudes about longer preservation time among surgeons and patients

� Meet anticipated greater demand for tissue with an aging population

� Respond to future threats to the donor pool related to increase domestic demand and emerging infections

23

Accomplishments with

4°C Preservation Media since 1974 to 2012

� Established local based eye banking system with strong community support

that has been enhanced by regional sharing systems meeting local, regional

and national needs at present

� Serving international needs when tissue needs are met domestically

with preservation time up to 14 days

� Elective basis for surgery

� Tissue safety and quality standards

� Growth of keratoplasties to over 46,000 in 2011 domestically and over

18,000 (28%) internationally according to the EBAA

Courtesy G. Rosenwasser

Donor/Preoperative and Operative Factors Influencing Endothelial Survival after Keratoplasty at 4˚C

Donor/Preoperative

� Method of storage

� Medium

� Death to enucleation and preservation

� Preservation time

� Donor age

� Donor cell density

� Cause of death

� Recipient age

� Recipient diagnosis

– Keratoconus

– Fuchs’ dystrophy

– Pseudophakic bullous keratopathy

– HSV keratitis

– Regraft

– Chemical burn

� Glaucoma

� Synechiae

Operative

� Procedure: PKP, EK (DSEK, DMEK)

� Lens status: aphakic, AC IOL, PC IOL

� Tube shunt

24

5-Year Graft Success Rates and cell loss in

the CDS/SMAS

� Donor Age > 66 years

86% clear grafts

75% cell loss

� Donor Age < 66 years

86% clear grafts

69% cell loss

10-12 year results coming

out next year

5-Year Specular Microscopy Ancillary StudyOther Major Findings

� Baseline cell count does not predict graft failure within usual donor cell count range.

� 6 month count predictive: 13% failed with a count of less than 1700 cells/mm2 vs. 2% with a count of 2500 cells/mm2 or higher.

� 15% of clear grafts at 5 yrs had ECD < 500 cells/mm2.

� Larger grafts (> 8mm) have a higher cell density at 5 years

� Female donors had less cell loss than male donors: 67% vs. 72%

Clear graft at 5 yrswith ECD 428 cells/mm2Donor—3207 cells/mm2

25

Donor/Preoperative and Operative Factors

Influencing Endothelial Function after Keratoplasty at 4˚C

Donor/Preoperative

� Method of storage

� Medium

� Death to enucleation and preservation

� Preservation time

� Donor age

� Donor cell density

� Cause of death

� Recipient age

� Recipient diagnosis

– Keratoconus

– Fuchs’ dystrophy

– Pseudophakic bullous keratopathy

– HSV keratitis

– Regraft

– Chemical burn

� Glaucoma

� Synechiae

Operative

� Procedure: PKP, EK (DSEK, DMEK)

� Lens status: aphakic, AC IOL, PC IOL

� Tube shunt

•Less than 10 years old

•In 2011, 47% of all USA corneal transplants were EK (EBAA)

Endothelial Keratoplasty: DSEK and DMEK

Game Changer

DSEK PKP

Courtesy of F. Price

26

% Cell Survival

0

20

40

60

80

100

baseline 6 m 1 y 2 y 3 y

Time after transplant

DSAEK

PKP

DSEK vs. SMAS PKP Endothelial Cell Loss

M Price et al Ophthalmology In press

Single center cohort:5-yr cell survival DSEK > PKP

% cell

survivalEK

PK

0

20

40

60

80

100

donor 1 year 2 years 3 years 4 years 5 years

DSEK

PKP

* Price et al, Ophthalmology 2011

27

Concluding Remarks� Great strides over the past nearly 40

years with preservation media at 4°C: A

Great American Story

� Excellent graft success for low to

moderate risk cases

� Time from death to surgery has been

extended out to 14 days but this remains

to be definitively proven

� However, at least for PKP long term cell

loss is comparable for short term storage

to intermediate preservation in media at

4°C

� Procedure dependent—long term

endothelial survival improved with

DSEK vs PKP

Concluding Remarks

� Can we improve long-term

endothelial survival with this

approach further—perhaps

not

� May require new operative

and postoperative

interventions with novel

delivery systems

– Endothelial

transplantation

– Growth factors

– Anti-apoptotic agents:

ROCK inhibitor

3207 cells/mm2

donor

428 cells/mm2

5 years post PKP Shigeru Kinoshita

Primary MonkeyCorneal Endothelial cells

PKP endo cell loss

Control ROCK inhibitor

28

Jonathan H. Lass, M.D.

Charles I Thomas Professor and Chairman

CWRU Department of Ophthalmology and Visual Sciences

Director, University Hospitals Eye Institute

Medical Director, Cornea Image Analysis Reading Center

THANK YOU and the EBAA