Effect of Corneal Preservation Time on Long-Term Graft Success
Phase: PHASE4
Conditions: Cornea Preservation Time, Endothelial Keratoplasty, Transplant Success, Endothelial Cell Density
Sponsor: Case Western Reserve University

Cornea Preservation Time Study; Version 4.0; June 10, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
Effect of Corneal Preservation Time on Long-Term Graft Success 
1 
Cornea Preservation Time Study (CPTS) 
2 
Protocol 
3 
 
4 
Version 4.0 
5 
July 1, 2015 
6 
 
7 
Study Chair: Jonathan Lass MD 
8 
Department of Ophthalmology & Visual Sciences 
9 
Case Western Reserve University 
10 
University Hospitals Eye Institute  
11 
11100 Euclid Ave; 4129 Lakeside 
12 
Cleveland, OH 44106 
13 
 
14 
Data Management and Analysis Center (DMAC): 
15 
Director: Allison Edwards MS 
16 
Jaeb Center for Health Research 
17 
15310 Amberly Drive, Suite 350 
18 
Tampa, FL  33647 
19 
 
20 
Coordinating Center (CC): 
21 
Director: Loretta Szczotka-Flynn OD, PhD 
22 
Department of Ophthalmology & Visual Sciences 
23 
Vision Research Coordinating Center 
24 
Case Western Reserve University 
25 
University Hospitals Eye Institute  
26 
11100 Euclid Ave; 4126 Lakeside 
27 
Cleveland, OH 44106 
28 
 
29 
Cornea Image Analysis Reading Center (CIARC): 
30 
Director: Beth Ann Benetz, M.A. 
31 
Department of Ophthalmology & Visual Sciences 
32 
Case Western Reserve University 
33 
University Hospitals Eye Institute 
34 
11100 Euclid Ave;  Wearn 646 
35 
Cleveland, OH 44106 
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Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
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TABLE OF CONTENTS 
40 
CHAPTER 1.  BACKGROUND AND RATIONALE 
4 
41 
1.1.  BACKGROUND 
4 
42 
1.1.1  STUDIES OF PRESERVATION TIME 
5 
43 
1.2.  RATIONALE 
6 
44 
1.3.   STUDY OBJECTIVES 
7 
45 
1.4.  SYNOPSIS OF STUDY DESIGN 
7 
46 
1.4.1.  STUDY DESIGN 
7 
47 
1.4.2.  MAJOR ELIGIBILITY CRITERIA 
7 
48 
1.4.2.1.  STUDY PARTICIPANTS 
7 
49 
1.4.2.2.  DONOR CORNEAS 
8 
50 
1.4.2.3.  TREATMENT GROUPS 
8 
51 
1.4.2.4.  SAMPLE SIZE 
8 
52 
1.4.2.5.  VISIT SCHEDULE AND PROCEDURES 
8 
53 
1.4.2.6.  OUTCOMES 
9 
54 
1.5.  GENERAL CONSIDERATIONS 
9 
55 
 
56 
CHAPTER 2.  ELIGIBILITY AND ENROLLMENT CLINICAL SITES 
10 
57 
2.1.  ELIGIBILITY ASSESSMENT 
10 
58 
2.2.  ELIGIBILITY CRITERIA 
10 
59 
2.2.1.  STUDY PARTICIPANT ELIGIBILITY CRITERIA 
10 
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2.2.1.1  STUDY PARTICIPANT INCLUSION CRITERIA 
10 
61 
2.2.1.2  STUDY PARTICIPANT EXCLUSION CRITERIA 
10 
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2.2.2.  STUDY EYE INCLUSION CRITERIA 
10 
63 
2.2.3.  STUDY EYE EXCLUSION CRITERIA 
11 
64 
2.3.  ELIGIBILITY CRITERIA FOR SECOND STUDY EYE 
11 
65 
2.4.  SCREENING EVALUATION AND BASELINE TESTING 
11 
66 
2.4.1.  HISTORICAL INFORMATION 
11 
67 
2.4.2.  BASELINE TESTING 
12 
68 
2.5.  SUBJECT ENROLLMENT 
12 
69 
2.6.  RANDOMIZATION 
12 
70 
 
71 
CHAPTER 3.  DONOR ELIGIBILITY AND CORNEA ASSIGNMENT 
72 
                         EYE BANKS 
13 
73 
 
74 
3.1.  EYE BANK PROCEDURES 
13 
75 
3.1.1.  DONOR ELIGIBILITY 
13 
76 
3.1.2.  ASSIGNMENT OF DONOR CORNEAS TO STUDY PARTICIPANTS 
13 
77 
                3.1.2.1.  DONOR NOT AVAILABLE 
14 
78 
                3.1.2.2.  RESCHEDULED SURGERY AND/OR REASSIGNMENT OF TISSUE 
14 
79 
3.1.3.  EYE BANK PROCEDURES FOR STUDY IMAGES 
14 
80 
3.1.3.1  SCREENING IMAGES 
14 
81 
3.1.3.2  PRE-OPERATIVE IMAGES 
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Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
2 
CHAPTER 4.  TRANSPLANTATION AND FOLLOW UP CLINICAL SITES 
15 
83 
4.1.  ENDOTHELIAL KERATOPLASTY PROCEDURE 
15 
84 
4.2.  POST-OPERATIVE MANAGEMENT 
15 
85 
4.3.  FOLLOW-UP VISIT SCHEDULE 
15 
86 
4.4.  TESTING PROCEDURES  
16 
87 
4.5.  DEFINITION OF TESTING PROCEDURES 
16 
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4.5.1.  SLIT LAMP EXAMINATION 
16 
89 
        4.5.1.1 RECIPIENT CORNEAL STROMA CLARITY 
16 
90 
        4.5.1.2  DONOR CORNEAL STROMA CLARITY 
17 
91 
        4.5.1.3  GRAFT REJECTION ASSESSMENT 
17 
92 
4.5.2.  INTRAOCULAR PRESSURE 
17 
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4.5.3.  ULTRASONIC PACHYMETRY 
17 
94 
4.5.4.  SPECULAR/ CONFOCAL MICROSCOPY 
17 
95 
4.6.  ADDITIONAL PROCEDURES 
17 
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4.7.  GRAFT FAILURE 
18 
97 
4.8.  FINAL STATUS 
19 
98 
CHAPTER  5.  ADVERSE EVENT REPORTING 
20 
99 
5.1.  ADVERSE EVENT REPORTING AND REVIEW 
20 
100 
5.2. DATA AND SAFETY MONITORING COMMITTEE REVIEW OF ADVERSE EVENTS 
21 
101 
5.3.  RISKS AND DISCOMFORTS 
21 
102 
CHAPTER 6.  MISCELLANEOUS CONSIDERATIONS 
23 
103 
6.1.  POTENTIAL BENEFITS TO SUBJECTS 
23 
104 
6.2.  ALTERNATIVE(S) TO PARTICIPATION 
23 
105 
6.3.  SPECIAL CONSIDERATIONS IN FOLLOW-UP 
23 
106 
6.4.  WOMEN AND MINORITIES 
23 
107 
6.5.  FINANCIAL INFORMATION 
24 
108 
6.6.  CONFIDENTIALITY 
24 
109 
6.7.  PRIVACY OF PROTECTED HEALTH INFORMATION 
24 
110 
 
111 
CHAPTER 7.  STATISTICAL METHODS 
25 
112 
 
113 
7.1.  SAMPLE SIZE 
25 
114 
7.2.  DATA ANALYSIS 
25 
115 
7.2.1.  PRIMARY ANALYSIS OF GRAFT FAILURE 
25 
116 
7.2.1.1.  UNADJUSTED ANALYSIS 
26 
117 
7.2.1.2.  ADJUSTED ANALYSIS 
26 
118 
7.2.2.  SECONDARY ANALYSES OF GRAFT FAILURE 
26 
119 
7.2.2.1.  PRESERVATION TIME 
26 
120 
7.2.2.2.  PREDICTIVE FACTORS 
27 
121 
7.2.2.3.  ECD AS TIME DEPENDENT PREDICTOR OF GRAFT FAILURE 
27 
122 
                7.2.3.  GRAFT REJECTION 
28 
123 
7.2.4.  ENDOTHELIAL CELL DENSITY (ECD) 
28 
124 
7.2.4.1.  INCLUDED SUBJECTS 
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Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
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7.2.4.2.  OUTCOME MEASURES 
28 
126 
7.2.4.3.  DESCRIPTIVE STATISTICS 
28 
127 
7.2.4.4.  PRIMARY ANALYSIS 
28 
128 
7.2.4.5.  SECONDARY ANALYSIS 
29 
129 
7.2.4.5.1.  SENSITIVITY ANALYISIS 
29 
130 
7.2.4.5.2.  ANALYSIS WITH DONOR TISSUE PRESERVATION TIME               
131 
                   AS CONTINUOUS/ MULTI-CATEGORICAL VARIABLE 
29 
132 
7.2.4.5.3.  LONGITUDINAL ANALYSIS 
29 
133 
7.2.5.  COURSE OF CORNEA CHANGES AFTER ENDOTHELIAL KERATOPLASTY 
29 
134 
7.2.6.  SAFETY ANALYSIS PLAN 
29 
135 
7.2.7.  ADDITIONAL TABULATION AND ANALYSES 
30 
136 
7.2.8.  DSMC INTERIM ANALYSIS PLAN 
30 
137 
 
138 
REFERENCES 
31 
139 
 
140 
APPENDIX A:    
141 
PROTOCOL AMENDMENT; EXTENSION OF FOLLOW-UP TO COMMON  END DATE 
33 
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1U10EY020797-01 A1 (A. Edwards PI) 
 
4 
Chapter 1 
144 
BACKGROUND AND RATIONALE 
145 
 
146 
1.1 
Background 
147 
The Cornea Donor Study (CDS) was conceived in 1997 when a real threat to the donor pool was 
148 
present based on the concerns of an emerging HIV and hepatitis epidemic, the impact of 
149 
refractive surgery procedures, an aging population with a rise in the number of Fuchs’ 
150 
Endothelial Corneal Dystrophy (FECD) and pseudophakic bullous keratopathy (PBK) cases in 
151 
the United States, and a worldwide demand for corneal tissue. This was most prescient, since the 
152 
number of corneas provided by eye banks in the United States subsequently rose from 43,492 
153 
cases in 1997 to 59,271 in 2010, a 37% increase in demand.(1)  This demand has been met by 
154 
our phenomenal success in changing the perception among patients, surgeons, and the eye bank 
155 
community that older donor tissue (> 65 years of age to 75) is as suitable and successful in 
156 
keratoplasty as younger tissue, at least for the management of FECD and PBK cases. The use of 
157 
older donor tissue has resulted in opening up a whole pool of tissue that would not have been 
158 
used by many surgeons in the United States.  This change in perception is the result of a simple, 
159 
but powerful, evidence-based prospective, masked clinical trial, which showed 86% graft success 
160 
following penetrating keratoplasty (PKP) for endothelial dysfunction conditions at 5 years in 
161 
both the ≥ 65 to 75 year donor group and the <65 donor group. As an aside, the CDS also 
162 
demonstrated the tremendous value of an academic and community network of well trained 
163 
corneal surgeons and their ability to work with a coordinating center and corneal endothelial 
164 
image analysis reading center to produce high quality and reliable data with a tremendous impact 
165 
on practice patterns in corneal transplantation. 
166 
 
167 
Several factors which had the potential to impact the donor supply in 1997 still remain, new 
168 
issues have arisen, and all impact the future efficient provision donor tissue and need for 
169 
increasing the donor supply: 
170 
 
171 
1. The donor pool is impacted by a continued threat from common viral infections, in particular 
172 
Hepatitis B, with 2,698 donors alone rejected in 2008 and 3,631 donors in 2010 (a 34% 
173 
increase in unusable tissue from the previous year) based on a positive Hepatitis B Core 
174 
(HBcAB) antibody test.(1) 
175 
2. With increasing FDA regulations towards testing for emerging infections, such as West Nile 
176 
Virus (from 6 cases in 2008 to 97 cases in 2010, a more than 15 fold increase,(1) and Chagas 
177 
Disease,(2.3) more tissue will be rejected, or delays in test results will result in cancellation 
178 
of transplants. In addition, as additional serologic testing is added, the test could delay tissue 
179 
release or result in false positives that could even decrease tissue supply. 
180 
3. For serious potentially transmissible diseases, such as Prion agents, no reliable laboratory test 
181 
currently exists and only historical screening can be used to safeguard transplant recipients. 
182 
This strategy is justified only as long as the general population prevalence rate of Prion 
183 
disease is small.  If testing for potential slow virus diseases disqualifies a substantial number 
184 
of donors, this could have a tremendous negative impact on the donor pool. 
185 
4. The impact of an expanding aging population over 65 with the addition of the baby boomers 
186 
will result in a greater number of FECD and PBK cases, and increased demand for tissue. 
187 
5. The phenomenal growth of endothelial keratoplasty (EK), specifically Descemet Stripping 
188 
Endothelial Keratoplasty (DSEK) or Descemet Stripping Automated Endothelial 
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1U10EY020797-01 A1 (A. Edwards PI) 
 
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Keratoplasty with the use of an automated microkeratome (DSAEK), in the past five years 
190 
with a more than three-fold increase from 6,027 cases in 2006 to 19,159 in 2010(1) has 
191 
opened up a new pool of patients in which endothelial dysfunction cases are being treated 
192 
surgically before structural damage occurs.(4)  This growth has been assisted by improved 
193 
efficiency as a result of an increasing number of eye banks preparing the donor tissue for the 
194 
EK procedure, rather than in the operating room by the surgeon.  At the same time, the 
195 
primary donor failure rate for EK has been reported as high as  5% on average(4) is 10x as 
196 
high as in PKP (e.g. in the CDS, reference 5). As this procedure gains even wider acceptance, 
197 
further impact on the donor pool will occur.  Although donors that have anterior scars or 
198 
have undergone refractive surgical procedures may now be used for EK,(6) this positive 
199 
impact on the donor pool is offset by the earlier implementation of a surgical approach with 
200 
EK and a higher primary donor failure rate. 
201 
6. More tissue will also be needed in the future due to a potentially higher % yield loss of tissue 
202 
during the process of tissue cutting either for DSAEK or new procedures still under 
203 
development, including Descemet Membrane Automated Endothelial Keratoplasty 
204 
(DMAEK) and Descemet Membrane Endothelial Keratoplasty (DMEK). 
205 
7. An increasing number of donors are being rejected because of cataract surgery incisions that 
206 
are too close to the central cornea.  As the use of older donor tissue from previous cataract 
207 
surgery patients becomes more commonplace, prior endothelial damage from these cataract 
208 
incision wounds will impact the use of the larger donor EK grafts which are commonly up to 
209 
9 mm in diameter (personal communication, Gerald Cole, Tissue Banks International). 
210 
8. Unlike Canada, where transplant recipients must be on a long waiting list, American 
211 
surgeons and their patients have been fortunate to generally have their transplant surgical 
212 
cases on a scheduled basis.  With all the issues above, cancelled surgeries due to the lack of 
213 
tissue could occur frequently, resulting in lost revenues and added cost to the facility, the 
214 
surgeon, and in some cases the patient and families who have taken off work for the 
215 
procedure. 
216 
 
217 
With all these concerns listed above, flexibility with the use of longer preserved corneas within 
218 
the FDA guidelines will help increase the donor tissue used domestically and help to obviate 
219 
these concerns as they may arise. 
220 
 
221 
The status of the national donor supply remains dynamic with fluctuations on a daily, weekly, 
222 
and monthly basis depending on donor, surgeon, and patient supply and demand.  It is these 
223 
dynamic fluctuations and the various threats that loom to the donor supply listed above, 
224 
including emerging infections, which have driven the eye banking community to continually find 
225 
better ways to have a more orderly donor tissue supply with maximum flexibility within FDA 
226 
limits for tissue usage.  This continuing desire has led to the strong reaffirmation of this proposal 
227 
by the eye bank and surgeon community to help assure that there will be an adequate supply of 
228 
transplantable donor tissue when the study would be completed and results publicized in 2016-
229 
17. 
230 
 
231 
1.1.1 Studies of Preservation Time 
232 
Most studies have examined either death to surgery or time from preservation in medium to 
233 
surgery (preservation time) as a possible confounding variable or potential factor influencing 
234 
corneal clarity without it being the primary variable of interest; thus, the clinical outcome of 
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1U10EY020797-01 A1 (A. Edwards PI) 
 
6 
graft success and its relation to preservation time has been unclear.  In fact, there have been no 
236 
prospective masked trials that have randomized donor groups on this basis. 
237 
 
238 
PKP Findings: 
239 
x 
Chang et al(7) and Abbott et al(8) indicate that death-to-surgery times were not positively 
240 
correlated with graft clarity following PKP.  
241 
x 
Wagoner et al(9) showed in a retrospective study of 234 PKPs utilizing donor tissue 
242 
ranging in preservation time from 168 to 348 hours in Optisol GS that the likelihood of graft 
243 
survival was not statistically significantly affected by progressively longer periods of donor 
244 
storage time with no primary donor failures. 
245 
x 
Doganay et al(10) with a group of 48 patients undergoing PKP for keratoconus, FECD, 
246 
and PBK, examined preservation time in one group up to an average of 233 hours in Optisol GS 
247 
(n=18) compared to another group on average of 21 hours.  No difference on graft survival was 
248 
noted in this small series. 
249 
x 
Sugar et al(11) noted that an increase in stromal edema and Descemet folds increased 
250 
with higher death-to-preservation time following PKP in the CDS, but death-to-surgery was not 
251 
a variable of interest and the time from death to use was limited to 5 days. 
252 
 
253 
EK Findings: 
254 
x 
Guttman(12) in a small, non-peer reviewed report, found greater cell loss at six months 
255 
following EK in those corneas that were used over 96 hours with the correlation lost at 12 
256 
months. The highest death to use time observed in the study was 182 hours (7.6 days).  
257 
x 
Price et al(13) showed with EK that ECD was not found to be significantly correlated 
258 
with death to preservation or death to use time.  
259 
x 
Chen et al(14) and Terry et al(15) found no influence of the time from death to 
260 
implantation on graft success following EK, but mean time was approximately 95 hours.  This 
261 
lack of correlation of storage time with graft success was also emphasized in an editorial by 
262 
Terry.(16) 
263 
x 
Terry et al(17) in 362 eyes following EK with storage time averaging 99 hours (range 21 
264 
to 186 hrs), showed no difference in cell loss at 2 years for those stored up to 4 days compared 
265 
to those stored up to 8 days. 
266 
 
267 
1.2 
Rationale 
268 
This study addresses an important public health issue related to the utilization of donor tissue for 
269 
corneal transplantation and the need to increase and secure the donor pool.  Similar to the bias 
270 
which existed regarding donor age prior to the initiation of the CDS, the majority of corneal 
271 
surgeons in the United States do not accept tissue with preservation time longer than 7 to 8 days, 
272 
even though the FDA approval of Optisol GS, since its introduction in the early 90’s, is for a 
273 
preservation time of up to 14 days.  Instead these corneas are exported to the international 
274 
community where they are routinely transplanted up to 14 days of storage.  A lack of 
275 
information, particularly on a preservation time over 7 days, has likely contributed to a bias 
276 
against using corneal tissue beyond this time.  This study will address this bias by examining two 
277 
parameters of long-term success: recipient corneal (stromal) clarity and endothelial cell density 
278 
following EK (4) for the endothelial dysfunction conditions that have moderate risk for failure, 
279 
FECD and PBK.  Demand for corneas has substantially increased with the advent of EK.(1)  In 
280 
addition to the increased demand, there are additional areas of concern which may impact the 
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1U10EY020797-01 A1 (A. Edwards PI) 
 
7 
future donor supply including potential changes in the cornea evaluation process such as 
282 
increased regulations and more extensive laboratory requirements to test for emerging infections, 
283 
e.g. Hepatitis B and C, West Nile Virus, Chagas Disease.(2.3)  By changing the practice pattern 
284 
and increasing utilization of tissue beyond 7 days up to the FDA approved, 14 days from 
285 
preservation to surgery, this will facilitate increasing the domestic donor supply enabling easier 
286 
distribution of tissue and more time for tissue evaluation to rule out emerging infections with 
287 
suspected donors. 
288 
 
289 
1.3 
Study Objectives 
290 
The primary objectives of the “Effect of Corneal Preservation Time on Long-Term Graft 
291 
Success” (CPTS) study are: 
292 
x To determine if the 3-year graft failure rate following EK performed with donor corneas with 
293 
a preservation time of 8 to14 days is non-inferior to the failure rate when donor corneas with 
294 
a preservation time of 7 or fewer days are used. 
295 
x To determine if the central corneal endothelial cell density 3-years after EK is related to 
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preservation time. 
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x To evaluate the effect of donor, operative and postoperative factors on graft failure and 
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endothelial cell density three years following EK. 
299 
 
300 
1.4 Synopsis of Study Design 
301 
 
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1.4.1. Study Design 
303 
The CPTS is a randomized, controlled clinical trial examining the impact of preservation time on 
304 
graft failure and endothelial cell density following EK. The study has been designed so that the 
305 
surgeons and eye banks can follow and provide their usual surgical and post-operative 
306 
procedures and care to study participants with the exception of assignment of donor tissue.  
307 
Study eyes will be randomly assigned to receive a donor cornea from preservation date to 
308 
surgery date of 8 to 14 days or a donor cornea from preservation date to surgery date of 7 or 
309 
fewer days, with a comparable death to preservation time for both groups. Surgeons and study 
310 
participants are masked to time from preservation to surgery. 
311 
 
312 
1.4.2. Major Eligibility Criteria 
313 
1.4.2.1. Study Participants 
314 
 
315 
Major eligibility criteria include:  
316 
x Study participant age between 30 and <91 years with a minimum life expectancy of 3 years 
317 
and at least one eligible eye 
318 
x Study eye is a candidate for EK due to one of two conditions related to endothelial 
319 
dysfunction:  
320 
o Fuchs’ Endothelial Corneal Dystrophy  (FECD) 
321 
o Aphakic/pseudophakic corneal edema 
322 
x Eyes with anterior chamber intraocular lens (IOL) are excluded 
323 
 
324 
Specific eligibility criteria are listed in Section 3.1.  The determination of eye eligibility is 
325 
performed at the time EK surgery is planned.  A participant can have two study eyes if both eyes 
326 
are eligible.  The eligibility of the second eye would be assessed at the time surgery on the 
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8 
second eye is being scheduled.  Surgery on the second study eye can be performed no sooner 
328 
than 6 weeks after EK on the first study eye. 
329 
 
330 
 
331 
1.4.2.2. Donor Corneas 
332 
Eye banks will follow their procedural routine for procurement of tissue and determining its 
333 
suitability for EK, including prior LASIK or photorefractive keratectomy donors, in accordance 
334 
with the Medical Standards and Procedure Manual of the EBAA(19,20).  This includes standard 
335 
serologic testing, specular microscopy, and slit lamp examination. 
336 
 
337 
The following major eligibility criteria will apply to all donor tissue assigned to study eyes: 
338 
x 
Meets current EBAA and eye bank standards for human transplantation 
339 
x 
Age of donor at time of death 10-75 years  
340 
x 
If the donor body was refrigerated or eyes on ice within 10 hours of death, the body or 
341 
 
eye may stay refrigerated up to <20 hrs; if no refrigeration then the death to preservation 
342 
 
time should be <10 hrs 
343 
x 
Eye bank determined minimum ECD of ≥ 2300 cells/mm2   (upon the initial screening 
344 
 
determination of ECD) 
345 
x 
Polymorphism/Polymegethism: None to no more than mild changes (slight) 
346 
x 
Guttae: no true guttae present  
347 
x 
No evidence of central endothelial cell damage/trauma or dystrophy, such as FECD 
348 
 
 
349 
1.4.2.3. Treatment Groups 
350 
Enrolled eyes of study participants will be randomly assigned to receive either a donor cornea 
351 
with a preservation time of 8 to 14 days or a donor cornea with a preservation time of 7 or fewer 
352 
days.  
353 
 
354 
1.4.2.4. Sample Size 
355 
1,330 study eyes from up to 1,330 study participants, depending on what proportion of 
356 
participants elect and are eligible to enroll both eyes. 
357 
 
358 
1.4.2.5. Visit Schedule and Procedures 
359 
Enrolled eyes of study participants will be examined at a baseline/enrollment visit, at the time of 
360 
EK surgery, and at post-operative visits at 1 day, 1 week, 1 month, 6, 12, 24, and 36 months in 
361 
addition to any routine care visits.  Participants with two study eyes will follow a modified visit 
362 
schedule to minimize return visits for participants that had bilateral EK as part of the study. 
363 
 
364 
Procedures at each protocol visit will follow the surgeon’s standard of care in addition to detailed 
365 
and standardized measurements of recipient and donor corneal stroma clarity, pachymetry, and 
366 
central endothelial cell density as outlined in the Table in Section 4.3.  Non-protocol visits will 
367 
follow the surgeon’s standard of care.  
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9 
1.4.2.6. Outcomes 
370 
Primary Outcome Measure: Graft failure, defined as the occurrence of one of the following 
371 
within 3 years of surgery: 
372 
x Regrafting of the study eye for any reason 
373 
x Cornea which remains cloudy without clearing, according to the following:    
374 
(1)  cloudy cornea on the first postoperative day which does not clear within 8 weeks    
375 
OR  
376 
(2) cloudy cornea which was initially clear postoperatively but becomes and remains 
377 
cloudy for 3 months without clearing.    
378 
Secondary Outcome Measure:  Endothelial cell density at 3 years from surgery, conditional on 
379 
graft survival at 3 years from surgery. 
380 
 
381 
1.5 General Considerations 
382 
The study is being conducted in accordance with the ethical principles that have their origin in 
383 
the Declaration of Helsinki, with the protocol described herein, and with the standards of Good 
384 
Clinical Practice.  The CPTS Procedures Manuals provide details of the procedures followed by 
385 
the eye banks and by the clinical sites. Data will be directly collected in electronic case report 
386 
forms, which will be considered the source data. 
387 
 
388 
389 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
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10 
Chapter 2 
390 
ELIGIBILITY AND ENROLLMENT 
391 
CLINICAL SITES 
392 
 
393 
2.1 
Eligibility Assessment 
394 
Eligibility is assessed during a routine examination by an investigator, as there are no 
395 
examination procedures required to assess patient eligibility other than those that are part of 
396 
standard patient care. 
397 
 
398 
A participant can have two study eyes if both eyes are eligible at the time of enrollment or if the 
399 
second eye becomes eligible at a later time.  The determination of eye eligibility is performed at 
400 
the time EK surgery is planned, meaning that the eligibility of the second eye will be assessed at 
401 
the time surgery on the second eye is being scheduled.  Surgery on the second eye can be 
402 
performed no sooner than 6 weeks after EK on the first eye.  Participants with two eligible eyes 
403 
will have the option of deciding whether to include one or both eyes in the study. 
404 
 
405 
2.2      Eligibility Criteria 
406 
To be eligible, a study participant must meet the participant inclusion criteria and have at least 
407 
one eye meeting the study eye inclusion criteria and none of the exclusion criteria. 
408 
 
409 
2.2.1. Study Participant Eligibility Criteria 
410 
2.2.1.1 Study Participant Inclusion Criteria 
411 
1) Age range between 30 and <91 years with minimum life expectancy of at least 3 years. 
412 
2) Willingness to return for follow-up study visits at 1 day, 1 week, 1 month, 6 months, 1 year, 
413 
2 years and 3 years. 
414 
3) Fluent in English or Spanish. 
415 
 
416 
2.2.1.2 Study Participant Exclusion Criteria 
417 
1) 
Decisionally and/or cognitively impaired 
418 
 
419 
2.2.2 Study Eye Inclusion Criteria 
420 
1) EK is scheduled between 10 and 60 days after enrollment 
421 
x The 10-day requirement relates to the need to be able to randomly assign the eye to 
422 
either intervention group. 
423 
x The 60-day requirement relates to the need to have current eligibility and enrollment 
424 
data at the time of surgery. If surgery is postponed to >60 days after the initial 
425 
enrollment visit, a new Baseline Visit and eligibility assessment will have to be 
426 
performed. 
427 
2) Presence of a condition related to endothelial dysfunction which will be treated by EK. 
428 
x Eligible indications for EK include: 
429 
a. Presence of  FECD meeting at least one of the following: 
430 
¾ Phakic FECD 
431 
¾ Phakic FECD with cataract 
432 
o Triple procedure including EK for FECD, cataract extraction and 
433 
posterior chamber intraocular lens implantation (IOL) is allowed 
434 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
11 
¾ Aphakic FECD 
435 
¾ Pseudophakic FECD with posterior capsule supported, suture-fixated, or 
436 
sulcus-supported posterior chamber IOL 
437 
b. Aphakic or pseudophakic corneal edema with posterior capsule supported, suture- 
438 
fixated, or sulcus-supported posterior chamber IOL without FECD 
439 
 
440 
2.2.3 Study Eye Exclusion Criteria 
441 
1) Prior EK 
442 
2) Indication for surgery that is not suitable for EK (e.g, keratoconus, stromal dystrophies and 
443 
scars) 
444 
3) Presence of a condition that has a very high probability for failure (e.g., failed EK or PKP, 
445 
heavily vascularized cornea, uncontrolled uveitis) 
446 
4) Other primary endothelial dysfunction conditions including posterior polymorphous corneal 
447 
dystrophy and congenital hereditary corneal dystrophy 
448 
5) Anterior chamber IOL in study eye prior to or anticipated during EK 
449 
6) Planned intraocular lens exchange of an anterior chamber IOL with a posterior chamber IOL 
450 
in study eye at time of study EK 
451 
7) Pre-operative central sub-epithelial or stromal scarring that the investigator believes is 
452 
visually significant and could impact post-operative stromal clarity assessment 
453 
8) Stromal vascularization that is visually significant (by investigator’s judgment) 
454 
9) Presence of anterior synechiae (iris to cornea) 
455 
10) Peripheral anterior synechiae (iris to angle) in the angle greater than a total of three clock 
456 
hours 
457 
11) Hypotony (Intraocular pressure <10 mm Hg) 
458 
12) Uncontrolled (defined as intraocular pressure > 25mm Hg) glaucoma with or without prior 
459 
filtering surgery or shunt or mini-shunt placement. 
460 
A shunt or mini-shunt is any device implanted to lower intraocular pressure through an external route 
461 
(e.g Ahmed) or internal route (e.g. Glaukos) that is present in the anterior chamber angle or extends into 
462 
the anterior chamber.   
463 
13) Controlled glaucoma with prior shunt or mini-shunt placement for glaucoma 
464 
o Note: FECD or pseudophakic/aphakic corneal edema with posterior chamber IOL that 
465 
also have undergone filtering surgery (without shunt or mini-shunt) in which 
466 
glaucoma is currently considered under control will be eligible 
467 
14) Fellow eye visual acuity < 20/200 that is not correctable with EK 
468 
 
469 
2.3 Eligibility Criteria for Second Study Eye 
470 
1) Study participant has already enrolled one eye 
471 
2) The second eye meets all study eye inclusion and exclusion criteria (2.2.2 and 2.2.3) 
472 
3) EK surgery in second eye is not planned within 6 weeks of EK on first study eye 
473 
 
474 
2.4 
Screening Evaluation and Baseline Testing 
475 
2.4.1 Historical Information 
476 
A history will be elicited from the potential study participant and extracted from available 
477 
medical records.  It is anticipated that potential participants will be patients within the practices 
478 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
12 
of the site investigator who are deciding to undergo EK with the respective surgeon.  Thus, 
479 
obtaining histories relevant to the CPTS eligibility criteria will be part of routine care. 
480 
 2.4.2 Baseline Testing 
481 
Potential eligibility will be assessed as part of a routine-care examination as stated above.  
482 
However, prior to completing any procedures or collecting any data that are not part of usual 
483 
care, written informed consent will be obtained.  
484 
In addition to the usual assessment for candidates for EK for the two acceptable conditions for 
485 
the CPTS, the surgeons will grade disease severity in those study participants who have FECD 
486 
employing the FECD Genetics Multi-center Study Grading Assessment Guide (18).  Family 
487 
history of FECD will be solicited.   
488 
2.5 Subject Enrollment 
489 
A maximum of 1330 participants will be enrolled, depending on the number of participants who 
490 
enroll both eyes into the study to reach the recruitment goal of 1330 study eyes with a goal to 
491 
enroll an appropriate representation of minorities.  As the enrollment goal approaches, sites will 
492 
be notified of the end date for recruitment.  Study participants who have signed an informed 
493 
consent form can be randomized up until the end date. 
494 
 
495 
After the informed consent form is signed, enrollment will be accomplished using the study 
496 
website.  Enrollment must be completed at least 10 days prior to the date of surgery. The study 
497 
participant is then managed according to the investigator’s usual routine without regard to the 
498 
fact that the study participant is participating in the trial. 
499 
2.6 Randomization 
500 
 
501 
Randomization of participant eyes will occur via an automated computer program. The 
502 
randomization schedule will be stratified by surgeon using a permuted blocks design.  The 
503 
randomization groups are as follows: 
504 
x Preservation Time Group:  <7 days 
505 
x Preservation Time Group:  8-14 days 
506 
 
507 
 
508 
A study participant may have both eyes enrolled in the study.  The eye scheduled for surgery 
509 
which will occur first will be assigned randomly to a preservation time group, and the eye 
510 
scheduled for surgery which will occur second will be assigned to the alternate group. 
511 
 
512 
 
 
513 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
13 
Chapter 3 
514 
DONOR ELIGIBILITY AND CORNEA ASSIGNMENT 
515 
EYE BANKS 
516 
 
517 
3.1 Eye Bank Procedures 
518 
With the exception of procedures related to assignment of a cornea to a participant eye, specular 
519 
microscopy external calibration and technician certification procedures and study procedures for 
520 
obtaining specular images, eye bank procedures will mimic standard procedures as closely as 
521 
possible to minimize disruption to their normal routine.  Eye banks will be able to use any FDA-
522 
approved media for intermediate term storage up to 14 days at 4oC.   
523 
 
524 
3.1.1 Donor Eligibility  
525 
All eye banks will follow their procedural routine for procurement of tissue and determination of 
526 
suitability for EK, including prior LASIK or PRK donors, in accordance with the Medical 
527 
Standards and Procedure Manual of the EBAA(19,20).  This includes standard serologic testing, 
528 
specular microscopy, and slit lamp examination. 
529 
 
530 
The following eligibility criteria will apply to all donor tissue assigned to participant eyes: 
531 
x Obtained from an EBAA accredited eye bank 
532 
x Meets current EBAA and eye bank standards for human transplantation 
533 
x Age of donor at time of death 10-75 years 
534 
x If the donor body was refrigerated or eyes on ice within 10 hours of death, the body or eye 
535 
may stay refrigerated up to <20 hrs; if no refrigeration then the death to preservation  time 
536 
should be <10 hrs 
537 
x Eye bank determined minimum ECD of ≥ 2300 cells/mm2   (upon the initial screening 
538 
determination of ECD) 
539 
x Polymorphism/Polymegethism: None to no more than mild changes (slight) 
540 
x Guttae: no true guttae present  
541 
x No evidence of central endothelial cell damage/trauma or dystrophy, such as FECD. 
542 
 
543 
3.1.2 Assignment of Donor to Study Eyes 
544 
The primary eye bank, as designated by the clinical site, will receive a notification when there is 
545 
a pending donor assignment. The   Eye Bank Procedures Manual provides details regarding the 
546 
assignment process.  When the eye bank submits an assignment request, a computer program 
547 
will use a minimization algorithm to evaluate all available donors and ensure a balance of 
548 
subgroups (0-4 days, 5-7 days, 8-11 days, 12-14 days from preservation to surgery) within the 
549 
primary randomization groups.  Time from preservation to scheduled surgery date will be 
550 
calculated as whole days for the purpose of assignment into the appropriate randomized 
551 
preservation time group.  As surgeons will be masked to all donor information, the label and 
552 
report that accompanies the donor will be generated from the study website in such a way to 
553 
maintain masking. 
554 
 
555 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
14 
After the donor assignment procedure is completed, the eye bank will complete a donor 
556 
information form on the study website, which includes information about the retrieval of the 
557 
cornea (date/time of death, date/time of retrieval, aspects of the processing), cause of death, age 
558 
of the donor, and ECD.  
559 
 
560 
For those assignments where pre-cut tissue has been requested, the eye bank will cut and prepare 
561 
the tissue on the same day that they would cut tissue if the donor was being assigned to a non-
562 
study participant for that particular surgeon, and will complete a cutting information form.   
563 
 
564 
3.1.2.1 Donor Not Available 
565 
If an eligible donor in the correct preservation   time window is not available at the primary eye 
566 
bank on the date of assignment, attempts will be made by the primary eye bank to import tissue 
567 
from another EBAA accredited eye bank.  If tissue in the correct preservation time window is 
568 
still not available, it will be up to the surgeon to either (1) reschedule the surgery or (2) inform 
569 
the participant that they will receive a donor that is otherwise healthy but does not meet criteria 
570 
to be in the study and that they will be discontinued from the study. 
571 
 
572 
3.1.2.2 Rescheduled Surgery and/or Reassignment of Tissue 
573 
If the surgeon rejects the assigned donor cornea for any reason, surgery will be rescheduled, and 
574 
a new assignment will be completed.    
575 
If surgery is rescheduled for any reason, and an already assigned donor cornea is no longer in the 
576 
correct preservation time window, a new assignment will be completed.    
577 
 
578 
3.1.3 Eye Bank Procedures for Study Images 
579 
Detailed procedures for obtaining best image quality and transmission to the DMAC will be 
580 
provided in the CPTS-CIARC Calibration, Certification, and Study Imaging Clinical Procedure 
581 
Manual and the technician(s) performing this procedure will be certified by the Cornea Image 
582 
Analysis Reading Center (CIARC).  
583 
 
584 
3.1.3.1 Screening Images 
585 
Up to 3 screening images of the central donor corneal endothelium obtained according to the  eye 
586 
bank’s usual procedure should be submitted to the DMAC.    
587 
 
588 
3.1.3.2 Pre-Operative Images 
589 
Three pre-operative images of the central donor corneal endothelium should be obtained in a 
590 
viewing chamber by a certified technician, and submitted to the DMAC. If the eye bank is 
591 
performing the cutting, the eye bank should obtain these pre-operative images after the tissue has 
592 
been cut.  If the surgeon is performing the cutting, the eye bank should obtain these pre-operative 
593 
images as close as possible prior to shipment to the surgeon following appropriate warming to 
594 
room temperature.   
595 
 
 
596 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
15 
Chapter 4 
597 
TRANSPLANTATION AND FOLLOW UP 
598 
CLINICAL SITES 
599 
 
600 
4.1 Endothelial keratoplasty procedure 
601 
Surgery (DSEK, DSAEK) will be performed according to the investigator’s usual routine.  
602 
Aspects of the surgical technique and procedure will be tracked, but not standardized.  Data to be 
603 
collected will include incision size, insertion method, air usage, other procedures (e.g. cataract 
604 
surgery), and operative complications (e.g., difficult donor preparation, difficult placement). As a 
605 
reminder, the DMAEK and DMEK procedures will not be acceptable endothelial keratoplasty 
606 
procedures for the CPTS nor will be PKP.    
607 
 
608 
The surgeon will be masked to donor parameters (e.g. donor age, donor ECD), except the FDA- 
609 
approved 40C preservation medium being employed (Optisol GS, Life 40C, etc.) and parameters 
610 
needed to perform the surgery (e.g. post-cut thickness).  Most importantly the surgeon and study 
611 
participant will be masked to preservation time. 
612 
 
613 
4.2 Post-operative Management 
614 
Postoperative management will be at the discretion of the surgeon based on his/her usual 
615 
practices.  Key aspects of pharmacologic management (e.g. topical corticosteroid usage) will be 
616 
collected on the data forms. 
617 
4.3 Follow-up visit schedule  
618 
Protocol-specified follow-up visits (and visit windows) for the first eye, established to conform 
619 
to the usual practice and timed from surgery date, will be as follows: 
620 
x Day 1 (1-2 days) 
621 
x Day 7 (5 – 9 days) 
622 
x Day 30 (20-40 days) 
623 
x 6 months (4-8 months) 
624 
x 12 months (10-14 months) 
625 
x 24 months (20 - 28 months) 
626 
x 36 months (35 – 42 months) 
627 
Additional visits can be performed more often at the discretion of the investigator.  A data form 
628 
will be completed for each protocol and non-protocol visit. For example, if graft failure is 
629 
determined and a regraft is required on a non-protocol visit, the appropriate follow-up visit form 
630 
and graft failure form should be completed if and when this occurs. 
631 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
16 
If the second eye of an active participant is enrolled, a modified visit schedule will be allowed 
632 
to minimize return visits for participants that had bilateral EK as part of the study. For example, 
633 
sites will be allowed to follow standard of care practices to avoid unnecessary visits linked to 
634 
targeting each eye within its respective windows as listed above.  Sites are encouraged to 
635 
schedule both eyes within their respective windows when the windows overlap, but at minimum 
636 
at least one eye must be within its target visit window at each visit. The only exception to this is 
637 
the 36 month visit which must be completed for each eye within its respective window, even it 
638 
an extra return visit is required. 
639 
4.4 Testing procedures 
640 
The Table below shows the key elements of data collection at each study visit.  Additional visits 
641 
may occur as needed for the usual care of the participant. 
642 
 
643 
 
Pre-
op 
1  
Day 
1 
 Week 
1 
 Month 
6  
Months 
12 
Months  
24 
Months 
36 
Months 
Parameters 
 
 
 
 
 
 
 
 
Medication History 
X 
X 
X 
X 
X 
X 
X 
X 
Slit lamp examination 
X 
X 
X 
X 
X 
X 
X 
X 
Intraocular pressure 
X 
 
X 
X 
X 
X 
X 
X 
Ultrasonic pachymetry 
 
 
X 
X 
X 
X 
X 
X 
Endothelial imaging 
 
 
 
 
X 
X 
X 
X 
Post-op complications and other 
untoward events  
 
X  
X  
X 
X 
X 
X 
X 
 
644 
4.5 Definition of testing procedures 
645 
4.5.1 Slit Lamp Examination 
646 
The slit lamp examination should be performed per the investigator’s usual routine. Specific 
647 
details of the data collected during the slit lamp examination are found in the site procedures 
648 
manual.   
649 
 
650 
4.5.1.1 Recipient corneal stroma clarity 
651 
The recipient corneal stroma clarity will be assessed by slit lamp examination using the 
652 
following 3-level classification:  
653 
 
654 
x clear central recipient stroma; 
655 
x equivocally cloudy central recipient stroma  
656 
x clouded central recipient stroma. 
657 
 
658 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
17 
Specific details regarding the grading of recipient corneal stroma clarity are found in the Site 
659 
Procedures Manual. Investigators will be provided a high resolution color standard scale and will 
660 
be trained and certified on this classification scheme prior to enrolling participants. 
661 
 
662 
4.5.1.2  Donor corneal stroma clarity 
663 
Donor corneal stroma clarity will be assessed by slit lamp examination. Specific details 
664 
regarding the grading of donor corneal stroma clarity are found in the Site Procedures Manual. 
665 
 
666 
4.5.1.3 Graft rejection assessment 
667 
Graft rejection will be assessed during the slit lamp examination using a modification of the 
668 
Collaborative Corneal Transplantation Studies (CCTS) classification (21,22). Graft rejection will 
669 
be classified as definite, probable/ possible, or not present. Details of the assessment of graft 
670 
rejection are found in the Site Procedures Manual.  
671 
The management of suspected graft rejection episodes will be according to the investigator 
672 
prerogative, but documented in the medication history. 
673 
 
674 
4.5.2 Intraocular pressure 
675 
Intraocular pressure will be measured using the investigator’s usual routine.   
676 
4.5.3 Ultrasonic pachymetry 
677 
Corneal thickness will be measured by a CPTS-provided ultrasonic pachymeter to ensure 
678 
standardization of this measurement across sites. Technical staff acquiring this measurement will 
679 
be trained and certified on study pachymeter use. If no measurement can be obtained (e.g. if the 
680 
cornea is too thick), this will be noted on the data form. Measurements taken on other 
681 
pachymeters will only be allowed if the study pachymeter is temporarily not functional.   
682 
4.5.4 Specular/confocal microscopy 
683 
Specular or confocal microscopy of the central endothelium will be obtained on all participants 
684 
that have not experienced graft failure to determine ECD by CIARC.  Detailed procedures for 
685 
obtaining best image quality and transmission to the DMAC will be provided in the CPTS-
686 
CIARC Calibration, Certification, and Study Imaging Clinical Procedure Manual and the 
687 
technician(s) performing this procedure will be certified by the CIARC. 
688 
4.6 Additional procedures 
689 
Data on all additional procedures performed on the study eye will be collected, including: 
690 
x air bubbling/repositioning in the first month 
691 
x cataract surgery and placement of intraocular lens (anterior chamber, posterior chamber)  
692 
x YAG capsulotomy  
693 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
18 
x refractive procedure (e.g. limbal relaxing incision, LASIK)  
694 
x glaucoma surgery (e.g. trabeculectomy, laser trabeculoplasty, tube shunt, mini-shunt, other)    
695 
Additionally, data on donor tissue rim cultures may be collected if performed as part of standard 
696 
of care. 
697 
 
698 
4.7 Graft Failure 
699 
Graft failure will be assessed and defined as the occurrence of one of the following: 
700 
x Cornea which requires regrafting for any reason 
701 
x Cornea which remains cloudy without clearing, according to the following:    
702 
(1)  cloudy cornea on the first postoperative day which does not clear within 8 weeks    
703 
OR  
704 
(2) cloudy cornea which was initially clear postoperatively but becomes and remains 
705 
cloudy for 3 months without clearing.    
706 
 A study participant whose cornea becomes cloudy (clouded recipient 
707 
central stroma, based on the modified CDS grading scale) will be treated 
708 
by the investigator’s usual routine.   
709 
For eyes meeting the definition of graft failure above, the principal cause of graft failure will be 
710 
classified as one of the following:  
711 
x Early failure (cloudy cornea on the first postoperative day which does not clear or 
712 
requires a regraft within 8 weeks), associated with surgical complications   
713 
x Primary donor failure (cloudy cornea on the first postoperative day which does not 
714 
clear or requires a regraft within 8 weeks),  in the absence of surgical complications   
715 
x Graft rejection (defined as a clouded recipient central stroma following an allograft 
716 
reaction);  
717 
x Non-rejection graft failure (defined as a graft that initially had a clear central recipient 
718 
stroma and becomes cloudy due to causes other than an immune event.  These include:  
719 
surface failure,  infection, glaucoma/hypotony, endothelial decompensation,  interface 
720 
irregularity or opacity, pre-existing stromal scarring, blunt or penetrating trauma, and 
721 
other causes);  
722 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
19 
x Refractive/visual graft failure (defined as a graft that requires regrafting due to 
723 
inadequate vision while the recipient central stroma remains clear).  
724 
 
725 
 
726 
4.8 Final Status 
727 
A Participant Final Status Form will be completed if the participant dies, withdraws, or is 
728 
deemed to be lost to follow-up by the CC staff.  An Eye Final Status Form will be completed if a 
729 
study eye is re-grafted, receives an AC IOL during surgery, experiences a suprachoroidal 
730 
hemorrhage during surgery,  receives a non-study donor cornea, will no longer have surgery, or 
731 
experiences enucleation, phthisis, graft failure due to blunt trauma, or graft failure due to 
732 
penetrating trauma,.  
733 
734 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
20 
CHAPTER 5 
735 
ADVERSE EVENT REPORTING 
736 
 
737 
 
738 
5.1 Adverse Event Reporting and Review 
739 
Adverse event information will be captured on the electronic case report forms or separate 
740 
adverse event forms completed after surgery and at all post-op visits.  Adverse events can be 
741 
systemic or ocular.  Death will be reported whether study related or not, with cause of death if 
742 
known; systemic events will be reported only if related or possibly related to study procedures.  
743 
Related and unrelated ocular adverse events will be reported only in the study eye as there is no 
744 
plausible reason to believe that the EK procedure could affect a non-study eye. 
745 
Each site will be responsible for informing the CC of any reportable adverse events as outlined in 
746 
the Site Procedures Manual.  The study chair will be responsible for abiding by reporting 
747 
requirements within the necessary time frames to the UHCMC IRB, NEI program officer, and 
748 
FDA, as required.  Each Principal Investigator is responsible for abiding by reporting 
749 
requirements specific to his/her IRB. 
750 
Certain adverse events may require expedited reporting.  Since this study does not involve 
751 
investigational drugs or devices and participants in this study would have undergone EK 
752 
regardless of study participation, expedited reporting of serious adverse events will be limited to 
753 
unanticipated and/or serious events in the study eye that are related or possibly related to 
754 
preservation time.  A list of events that require expedited reporting was determined in 
755 
conjunction with the DSMC.  The following events require separate adverse form completion 
756 
and expedited reporting by the site to the CC within 1 working day of learning of the event, and 
757 
then subsequently by the CC to the Medical Monitor on the same working day of notification and 
758 
to the NEI Program Office and designated DSMC member(s) within 1 week of notification: 
759 
x Endophthalmitis  
760 
x Microbial keratitis (bacterial, fungal, parasitic) within 3 months of EK 
761 
x Other unexpected, serious adverse events related or possibly related to 
762 
preservation time 
763 
 
764 
Operative and post-operative complications and all other adverse ocular findings will be 
765 
recorded on the case report forms and tabulated in semi-annual DSMC reports. Adverse events 
766 
presumed related to preservation time, study follow-up procedures of specular microscopy and 
767 
pachymetry, or systemic events related to EK will be captured on separate adverse event forms.  
768 
The DSMC will be provided the expedited adverse event reports and the tabulated semi-annual 
769 
reports in a manner that will enable them to unmask the treatment group if desired.   
770 
 
771 
  
772 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
21 
5.2 Data and Safety Monitoring Committee Review of Adverse Events 
773 
The DSMC has approved the protocol and template informed consent form; they will also 
774 
approve substantive amendments and will provide independent monitoring of adverse events.  
775 
Cumulative adverse event data are semi-annually tabulated for review by the DSMC.  Following 
776 
each DSMC data review, a summary will be provided to IRBs.  A list of specific adverse events 
777 
to be reported to the DSMC expeditiously is given in section 5.1  
778 
 
779 
5.3 Risks and Discomforts 
780 
The risks and discomforts for patients undergoing EK are the same regardless of study 
781 
participation.  Potential risks include: 
782 
x mild pain for approximately one week after surgery. 
783 
x temporary discomfort from the eye examination or eye drops, which may include stinging, 
784 
itching, or redness. 
785 
x serious infection or bleeding in 1 in 1,000 patients and serious problems related to anesthesia 
786 
in 1 in 10,000. 
787 
x in rare instances the topical drops can cause an allergic reaction, seizures, and an irregular 
788 
heartbeat. 
789 
x other potential risks include developing glaucoma, additional surgery due to healing 
790 
problems or movement out of position of the donor cornea, retinal swelling or detachment, or 
791 
loss of vision.  
792 
x rejection reactions occur approximately 10% of the time (23), but are usually reversible if 
793 
treated promptly with topical corticosteroids, but sometimes it leads to failure of the 
794 
transplant. 
795 
x measurement of intraocular pressure involves a topical anesthetic and fluorescein dye carries 
796 
a small risk of corneal abrasion and temporary corneal discomfort.  There is the rare 
797 
possibility of allergic reaction to the dye or anesthetic drops. 
798 
x Other risks of EK include: 
799 
o Endophthalmitis: a serious infection inside the eye that needs prompt treatment and may 
800 
cause permanent loss of vision or in severe circumstances loss of the eye 
801 
o Corneal infection:  a serious microbial infection of the cornea that requires immediate 
802 
treatment and may result in permanent scarring and possible permanent loss of vision 
803 
requiring a repeat of the corneal transplant 
804 
o Rare chance of dissemination of a communicable disease from the donor tissue 
805 
o Corneal scarring: permanent haze or cloudiness in the cornea that may result in 
806 
permanent loss of vision requiring a repeat of the corneal transplant 
807 
o Corneal neovascularization: blood vessel growth into the cornea that could subject the  
808 
transplant to a higher risk for rejection and/or permanent loss of vision, requiring a repeat 
809 
of the corneal transplant 
810 
o Corneal swelling: thickening of the cornea that may result in loss of vision which may or 
811 
may not be reversible.  If not reversible, another corneal transplant may be required to 
812 
restore the vision. 
813 
o Wrinkling of the corneal layers:  Wrinkling of the donor cornea as it heals may result in 
814 
blurred vision and require another corneal transplant. 
815 
 
816 
The following are risks of procedures that are not necessarily part of routine care but are 
817 
being performed for the purposes of this study: 
818 

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1U10EY020797-01 A1 (A. Edwards PI) 
 
22 
 
819 
o The anesthetics or instruments that touch the eye to check corneal thickness 
820 
(pachymeter) or image the endothelium (confocal or specular microscope) could 
821 
cause minor irritation and rarely breakdown of the surface corneal cells.  There is 
822 
the rare possibility of allergic reaction to the anesthetic drops or feeling faint from 
823 
the procedure. 
824 
 
825 
 
 
826 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
23 
CHAPTER 6 
827 
MISCELLANEOUS CONSIDERATIONS  
828 
 
829 
6.1 Potential Benefits to Subjects 
830 
Study participants will not benefit directly from participation in this study.  If longer preservation 
831 
time up to the FDA limit of 14 days can be shown to not adversely impact graft success and 
832 
endothelial cell density at 3 years, more donor tissue will be available for efficient distribution 
833 
within the United States for all keratoplasty procedures. In the future, if a study participant 
834 
requires another EK in either the same eye or their other eye, the information obtained from this 
835 
study might benefit them. 
836 
 
837 
6.2 Alternative(s) to Participation 
838 
Because of the nature of the study, the only other alternative is to not participate in the study. 
839 
The potential participants’ standing with his or her physician and/or hospital will not change. 
840 
 
841 
6.3 Special Considerations in Follow-up 
842 
In a long-term trial such as this in which outcome is not assessed for several years after 
843 
enrollment, special measures are necessary to assure that the participants will remain in follow 
844 
up and return for the outcome assessment examination.  Detailed contact information will be 
845 
collected at the time of enrollment and updated regularly. 
846 
The Coordinating Center will maintain contact with each patient.  Permission for such contacts 
847 
will be included in the Informed Consent Form.  The principal purpose of the contacts will be to 
848 
develop and maintain rapport with the participant and to update contact information.  The initial 
849 
phone contact will occur about one month following the EK surgery.  Subsequent phone contacts 
850 
will occur on a semi-annual basis.   Based on the experience with these calls by the Jaeb Center 
851 
in the CDS, the CC will similarly maintain this type of contact with participants throughout their 
852 
3 year time in the study which the CC and DMAC believe is critically important for the validity 
853 
of the study.  The purpose of these calls is not to collect study data to be used for monitoring or 
854 
in analyses or to provide medical information. Nor is it intended to schedule the participants for 
855 
their study visits; that is up to the local study coordinator.  This plan has been reviewed by the 
856 
IRB at UH Case Medical Center and felt to be feasible as long as incorporated into the consent 
857 
form at all our IRBs monitoring the study. 
858 
For participants who move out of the area of their study physician or whose medical insurance 
859 
coverage changes, an attempt will be made to have their care transferred to another study 
860 
physician.  When this is not possible, the CC will locate an ophthalmologist in the participant’s 
861 
new area to arrange for follow-up and the participant will be asked to sign a medical record 
862 
release form to provide the ophthalmologist with information as well as to obtain the results of 
863 
examinations performed by the ophthalmologist. 
864 
6.4 
Women and Minorities 
865 
We anticipate that study enrollment will be representative of the U.S. population of subjects who 
866 
undergo corneal transplantation for these endothelial conditions. Both males and females are 
867 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
24 
enrolled into each protocol. All ethnic and racial groups are eligible for participation in this 
868 
study, with the goal of having appropriate minority representation of those that undergo corneal 
869 
transplantation in the United States. 
870 
 
871 
6.5 
Financial Information 
872 
All visits, including but not limited to, pre-operative, post-operative, surgery, and any standard of 
873 
care follow-up appointments will be charged to the participant or his/her insurance carrier. The 
874 
participant will be responsible for any deductible or co-payments as defined by their particular 
875 
insurance carrier.  
876 
 
877 
The costs for pachymetry, and specular or confocal microscopy are considered research and the 
878 
costs will not be incurred by the participant. 
879 
 
880 
Study participants will be given a reimbursement of $25 for each study visit for travel costs. This 
881 
payment will be processed by the Jaeb Center. 
882 
 
883 
6.6    Confidentiality  
884 
The investigators will maintain the highest degree of confidentiality permitted for the clinical 
885 
and research information obtained from participants in this clinical study. Medical and research 
886 
records will be maintained in the strictest confidence. However, as part of the quality assurance 
887 
and legal responsibilities of an investigator, the site must permit authorized representatives of the 
888 
CC to examine (and when permitted or required by applicable law, to copy) clinical records for 
889 
the purposes of quality assurance reviews, audits, and evaluation of the study safety and 
890 
progress. Unless required by law, no copying of records with personally identifying information 
891 
will be permitted. Only the coded identity associated with documents or other participant data 
892 
may be copied (obscuring any personally identifying information) or transmitted to the CC. 
893 
Authorized representatives as noted are bound to maintain strict confidentiality of medical and 
894 
research information that may be linked to identified individuals. 
895 
 
896 
6.7   Privacy of Protected Health Information  
897 
The Health Insurance Portability & Accountability Act (HIPAA) is a Federal law that helps to 
898 
protect the privacy of the study participant’s health information and to whom this information 
899 
may be shared.  The Authorization forms used for this research study will tell the study 
900 
participant what health information (called Protected Health Information or PHI) will be 
901 
collected for this research study, who will see the study participant’s PHI and in what ways they 
902 
can use the information.  The researchers and staff must agree to protect the study participant’s 
903 
health information by using and disclosing it only as permitted by the subject in their 
904 
Authorization and as directed by state and Federal law.   
905 
 
 
906 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
25 
Chapter 7 
907 
STATISTICAL METHODS 
908 
 
909 
The approach to sample size and statistical analyses are summarized below.  A detailed statistical 
910 
analysis plan will be written and finalized prior to the completion of the study.  The analysis plan 
911 
synopsis in this chapter contains the framework of the anticipated final analysis plan.  
912 
 
913 
7.1 Sample Size 
914 
The sample size of 1330 is calculated based on a non-inferiority (a single one-sided test) design 
915 
with the goal to determine that the graft failure rate of the recipients of donor tissue transplanted 
916 
8 to 14 days after preservation is not worse than the graft failure rate of recipients of donor tissue 
917 
transplanted < 7 days after preservation. 
918 
 
919 
Non-Inferiority 
Limit 
Power = 90% 
Failure Rate 
12% 
10% 
8%a 
6% 
4% 
10% 
362 
310 
 
 
 
8% 
566 
482 
394 
 
 
6% 
1006 
858 
702 
538 
 
4 % 
2262 
1928 
1576 
1208 
824 
2% 
9044 
7708 
6304 
4832 3290 
Note: Numbers in table are total sample size for both treatment groups combined 
920 
(crossover and lost to follow up are not accounted for). Half would be randomized 
921 
to each group. 
922 
a CDS data: 3-year failure rate 
923 
 
924 
In the Cornea Donor Study (CDS), the 3-year failure rate was 8%.  Clinical expectations suggest 
925 
that the graft failure rate from the EK procedure will be smaller than the failure rate from the 
926 
PKP procedure that was used in CDS; therefore, a 3-year failure rate of 6% has been assumed.  
927 
Based on equal allocation of recipients to each group and type I error of 5%, a sample size of 
928 
1208 will provide 90% power for a non-inferiority limit of 4%.  Based on information from the 
929 
CDS, approximately 10% of subjects will have incomplete follow up (due to death, withdrawal 
930 
or lost to follow up) by the end of year 3.  Increasing the calculated sample size by this amount 
931 
gives a total of 1330 subjects (665 per group). 
932 
x The inclusion of participants with two study eyes, one in each treatment group, will tend 
933 
to reduce the variance and as a result increase statistical power. To be conservative, this 
934 
has not been accounted for in the sample size estimation, since the correlation of outcome 
935 
with two eyes is not known. 
936 
 
937 
7.2 Data Analysis 
938 
7.2.1 Primary Analysis of Graft Failure 
939 
Participant study eyes that did not have surgery, received a non-study donor, had an AC IOL 
940 
implanted during surgery, or experienced a suprachoroidal hemorrhage will be excluded from the 
941 
primary analysis. It is highly unlikely these events could be related to preservation time, thus 
942 
inclusion of them could actually bias towards concluding non-inferiority. Therefore, the primary 
943 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
26 
analysis will deviate from the principle of intent-to-treat, but this approach is conservative for a 
944 
non-inferiority analysis.   
945 
 
946 
In addition, the following will be censored at the last visit prior to occurrence: lost to follow-up, 
947 
withdrawn from study, death, enucleation, phthisis, or graft failures due to a blunt or penetrating 
948 
trauma. 
949 
 
950 
7.2.1.1 Unadjusted Analysis 
951 
     
952 
x Three year Kaplan-Meier graft failure estimates with 95% confidence intervals (variance 
953 
estimated using the Greenwood method) will be calculated separately for the two 
954 
treatment groups (≤7 days and 8 to 14 days from preservation to surgery). A one-sided 
955 
95% confidence interval will be constructed for the difference in 3 year graft failure rates 
956 
between the two groups. The bootstrap re-sampling technique will be used to account for 
957 
potentially correlated data from donors who donated both corneas in this study and 
958 
potentially correlated data from 2 study eyes of the same study participant.  The two 
959 
treatment groups will be declared equivalent if the one-sided 95% confidence interval for 
960 
the difference in proportions excludes the pre-defined non-inferiority limit of 4%. 
961 
 
962 
7.2.1.2 Adjusted Analysis 
963 
x Multivariate analysis will be performed using Cox proportional hazards regression model.  
964 
The primary multivariate model will include the corneal diagnosis regardless of statistical 
965 
significance, in addition to time from preservation to surgery.  In additional models, 
966 
potential confounders including recipient and donor age, recipient and donor race, presence 
967 
of glaucoma, presence of corneal vessels, history of smoking, and certain aspects of the 
968 
retrieval and processing of the donor tissue (including multiple types of storage media, if 
969 
more than one is used in preservation of the corneal tissues) will be screened by assessing 
970 
the change in the preservation time effect when the potential confounder is controlled for in 
971 
the Cox model. Variables that do not contribute significantly (P > 0.05) will be removed 
972 
from the model. 
973 
x Random surgeon effects will be tested using a generalized linear model via the SAS 
974 
GLIMMIX procedure.  This marginal model produces a robust standard error (RSE) by use 
975 
of a sandwich estimator, which corrects for correlated data. 
976 
x Potential effect modifiers of donor tissue preservation time such as recipient age or corneal 
977 
diagnosis will be screened by including first-order interaction terms.  Variables that exhibit 
978 
modification of the donor tissue preservation time effect with an associated P value < 0.05 
979 
will be retained in the model. 
980 
 
981 
 
982 
7.2.2 Secondary Analyses of Graft Failure 
983 
7.2.2.1 Preservation Time 
984 
The time from preservation to surgery is treated as a binary variable in the primary analysis (see 
985 
above section).  Secondary analyses will look at the time from preservation to surgery as a 
986 
categorical variable with multiple levels and as a continuous variable: 
987 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
27 
x Kaplan-Meier estimates of graft failure with 95% confidence interval will be calculated for 
988 
each of the following donor groups: 0 – 4, 5 – 7, 8 – 11, and 12 – 14 days from preservation 
989 
to surgery(counting partial days as whole days).   
990 
 
991 
x A Cox proportional hazards model will be constructed treating the time from preservation to 
992 
surgery as a continuous variable (using time of day to calculate hours from preservation to 
993 
surgery).  Polynomial terms will be added to assess any curvilinear, J, or U shaped 
994 
relationship between time from preservation to surgery and graft failure.  If no significant 
995 
departure from a linear relation is detected, a one sided 95% confidence interval will be 
996 
computed for the hazard ratio per day of time from preservation to surgery. 
997 
 
998 
x The proportional hazards assumptions will be tested through the use of time-dependent 
999 
variables with a logarithm transformation of time.  If this assumption is violated then hazard 
1000 
ratios will be presented separately for different periods following transplant. 
1001 
 
1002 
7.2.2.2 Predictive Factors 
1003 
A Cox model will be constructed including preservation time group regardless of statistical 
1004 
significance. Additional recipient/donor factors (see some examples listed below) will be 
1005 
considered for the model and included, if significantly associated with graft failure (p< 0.05). 
1006 
The proportional hazards assumptions will be tested as described above. 
1007 
 
1008 
x Recipient factors 
1009 
¾ preoperative diagnosis 
1010 
¾ gender 
1011 
¾ age 
1012 
¾ race  
1013 
¾ prior use of glaucoma medication 
1014 
¾ prior glaucoma surgery (trabeculectomy, laser procedure) 
1015 
¾ current smoker (at time of surgery) 
1016 
¾ lens status (phakic, posterior chamber intraocular lens) 
1017 
¾ Intraocular pressure (IOP) treated as a binary variable: < 25 vs. ≥ 25 mmHg 
1018 
 
1019 
x Donor/graft factors 
1020 
¾ 
eye bank determined screening ECD  
1021 
¾ 
pre-operative CIARC determined ECD 
1022 
¾ 
age 
1023 
¾ 
gender 
1024 
¾ 
race 
1025 
¾ 
history of diabetes 
1026 
¾ 
cause of death 
1027 
¾ 
type of storage medium 
1028 
 
1029 
7.2.2.3 ECD as Time Dependent Predictor of Graft Failure 
1030 
The relationship between endothelial graft failure (graft failure due to endothelial 
1031 
decompensation) and ECD will be addressed paralleling the methods used in the CDS.  A Cox 
1032 
model will be fit with ECD as a time dependant covariate. This analysis will be limited to 
1033 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
28 
subjects with at least one gradable follow up image.  The rate of change will also be calculated as 
1034 
a time dependent variable defined as the least squares slope over all previous measurements 
1035 
starting at 6 months (e.g., the rate of change at one year would be the slope fit to the 6 month and 
1036 
1 year ECD values).  Missing values will be imputed by Rubin’s method.  If significant departure 
1037 
from linearity is detected, then ECD will be treated as a categorical variable.  The proportional 
1038 
hazards assumptions will be tested as described above. 
1039 
 
1040 
To check whether results are sensitive to how missing data are handled, a second model will be 
1041 
fit with a time dependent indicator for missing ECD. 
1042 
 
1043 
7.2.3 Graft Rejection 
1044 
Associations of baseline recipient and donor factors with the occurrence of a graft rejection will 
1045 
be assessed in univariate and multivariate proportional hazards models. Life-table analyses will 
1046 
be used to compute the probability of a first rejection event within intervals defined by the study 
1047 
exam schedule.  Data will be censored at the time of a non-rejection graft failure or at the last 
1048 
visit.   
1049 
 
1050 
7.2.4 Endothelial Cell Density (ECD) 
1051 
7.2.4.1 Included Subjects 
1052 
The primary analysis will include all study participants with a gradable 3-year image, who have 
1053 
not experienced graft failure 3 years after transplantation. Study participants with a missing cell 
1054 
count at 3 years will be included in a secondary analysis using Rubin’s method of multiple 
1055 
imputation. 
1056 
 
1057 
7.2.4.2 Outcome Measures 
1058 
The primary outcome will be the ECD at 3 years, conditional on graft survival at 3 years. All 
1059 
other ECD measurements during follow up will be considered as a secondary outcome. 
1060 
 
1061 
7.2.4.3 Descriptive Statistics 
1062 
x Summary statistics (mean ± SD and/or median/quartiles as appropriate to the distribution) 
1063 
will be given for the ECD by the 2 treatment groups (≤7 and 8 to 14 days) and 4 treatment 
1064 
groups (0 – 4, 5 – 7, 8 – 11, and 12 – 14 days). 
1065 
x Change from eye bank determined screening ECD will be summarized in a similar manner. 
1066 
x Boxplots of ECD and change from eye bank determined screening ECD will be given for the 
1067 
2 randomization groups.   
1068 
x A scatter plot will be constructed of eye bank determined screening ECD vs. 3 year ECD 
1069 
with a symbol used to denote the two randomization groups. 
1070 
 
1071 
7.2.4.4 Primary Analysis 
1072 
The primary analysis will be limited to subjects with gradable 3 year images, who have not 
1073 
experienced graft failure 3 years after transplantation. An ANCOVA model with 3 year ECD as 
1074 
the dependent variable adjusting for eye-bank-determined screening ECD will be used to assess 
1075 
the effect of preservation time.  The time from preservation to surgery will be treated as a binary 
1076 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
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29 
variable. If residual values from the models above are highly skewed then a transformation (e.g., 
1077 
square root or logarithm) or non-parametric methods will be used instead. 
1078 
x Random effects will be modeled to account for any correlated data from the same donor and 
1079 
any correlated data from 2 study eyes of the same study participant. 
1080 
x Additional ANCOVA models will also adjust for other recipient/donor risk factors, (if p< 
1081 
0.05.  Random surgeon effects will also be explored using a mixed effects model.  
1082 
 
1083 
7.2.4.5 Secondary Analyses 
1084 
7.2.4.5.1 Sensitivity Analysis 
1085 
Sensitivity analysis will also be performed to check whether results change meaningfully 
1086 
depending on how missing data are handled. The missing 3 year ECD values for subjects with 
1087 
surviving grafts at 3 years will be imputed and included in an analysis as described in the 
1088 
previous section. The data imputation will be performed by using Rubin’s method of multiple 
1089 
imputation.  
1090 
 
1091 
 
1092 
7.2.4.5.2 Analysis with donor tissue preservation time as continuous/multi-categorical 
1093 
variable 
1094 
The analyses described above will be repeated with time from preservation to surgery treated as 
1095 
continuous (using time of day to calculate hours from preservation to surgery) or multi-category 
1096 
variable (0 – 4, 5 – 7, 8 – 11, and 12 – 14 days from preservation to surgery) in separate models. 
1097 
 
1098 
7.2.4.5.3 Longitudinal analysis 
1099 
This analysis also will be limited to subjects with a surviving graft at 3 years. A repeated 
1100 
measures least squares regression model will be fit using all available images at baseline, 6 
1101 
months, 1, 2, and 3 years. This analysis will be performed with and without imputation of 
1102 
missing data. Rubin’s method of data imputation will be used to impute the ECD values for all 
1103 
missing time points. The time from preservation to surgery will be modeled as both continuous 
1104 
and categorical as described above. If residual values have a skewed distribution then 
1105 
transformation (e.g., square root or logarithm) or non-parametric analysis will be used. 
1106 
 
1107 
7.2.5 Course of Cornea Changes After Endothelial Keratoplasty 
1108 
The association of donor, operative and postoperative related factors with ECD will be evaluated 
1109 
and assessed in univariate and multivariate ANCOVA models , adjusting for the reading center 
1110 
grading of pre-operative ECD (imaged post-cut if the eye bank was performing the cutting and 
1111 
imaged just prior to shipping if the surgeon was performing the cutting).  This ECD value will be 
1112 
considered the baseline for these analyses. 
1113 
 
1114 
7.2.6 Safety Analysis Plan 
1115 
The main safety analysis will involve tabulation of data by treatment group of events that could 
1116 
be considered possibly related to the preservation time such as post-op infection.  The efficacy 
1117 
analyses related to graft failure, corneal thickness, and ECDs also could be viewed as safety 
1118 
analyses.  Operative and post-operative complications will also be tabulated.  
1119 

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30 
 
1120 
Further details of the analytic approach will be provided in the detailed statistical analysis plan. 
1121 
7.2.7 Additional Tabulations and Analyses 
1122 
The following will be tabulated according to treatment group: 
1123 
x Baseline demographic and clinical characteristics 
1124 
x Visit completion rate for each visit 
1125 
x Protocol deviations 
1126 
  
1127 
7.2.8  DSMC Interim Analysis Plan  
1128 
No formal interim analyses are planned towards demonstrating non-inferiority before the end of 
1129 
the study since the recruitment period is planned to be short compared with the follow-up period 
1130 
and since we believe it is imperative to have three years of follow up to assess non-inferiority.   
1131 
. 
1132 
In addition to semi-annual review described in Section 5.2, the following plan for interim 
1133 
monitoring for a potential recommendation of early stopping of enrollment has been established 
1134 
in conjunction with the DSMC.  This plan is based on early donor failure rate and on the 
1135 
progress of recruitment.  
1136 
x Rate of failure within the first 8 weeks:  Upon enrollment of the first 100 eyes, and then 
1137 
quarterly thereafter (i.e., one review between each DSMC semi-annual meeting) the 
1138 
DMAC will evaluate the failure rate within the first 8 weeks (i.e. both the early failures 
1139 
and the primary donor failures, as defined in Section 4.6) in each group and notify the 
1140 
DSMC who will have the option of requesting additional information between the semi-
1141 
annual reviews.  The DSMC may also request more frequent reviews at any time.  
1142 
x Recruitment Progress:   Recruitment progress will be evaluated at the first two DSMC 
1143 
meetings following initiation of recruitment.    If based on the current recruitment total 
1144 
and recruitment trend over the previous 3 months, the projected timeline for the 
1145 
remaining recruitment is more than 16 months at the 1st review or more than 12 months at 
1146 
the 2nd review, the DSMC will discuss whether the study timeline can be met.  
1147 
Following each DSMC data review, a summary will be provided to the IRBs. 
1148 
 
1149 
 
1150 
 
1151 
 
1152 
 
1153 
 
1154 
1155 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
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31 
REFERENCES 
1156 
 
1157 
 
1158 
1.    Eye Bank Association of America. 2010 Eye Banking Statistical Report. 2010. 
1159 
2.    EBAA Regulatory Alert.  EBAA Comments on the U.S. Food and Drug Administration 
1160 
(FDA) Draft Guidance for Industry, (2009). 
1161 
3. Guidance for Industry.  Use of Nucleic acid tests to reduct the risk of transmission of West 
1162 
Nile Virus from donors of whole blood and blood components intended for transfusion and 
1163 
donors of human cells, tissues, and cellular and tissue-based products, (2008). 
1164 
4.   Lee WB, Jacobs DS, Musch DC, Kaufman SC, Reinhart WJ, Shtein RM. Descemet's 
1165 
stripping endothelial keratoplasty: safety and outcomes: a report by the American Academy 
1166 
of Ophthalmology. Ophthalmology. 2009;116(9):1818-30. 
1167 
5.  Gal RL, Dontchev M, Beck RW, Mannis MJ, Holland EJ, Kollman C, et al. The effect of 
1168 
donor age on corneal transplantation outcome results of the cornea donor study. 
1169 
Ophthalmology. 2008;115(4):620-6. 
1170 
6.  Armour RL, Ousley PJ, Wall J, Hoar K, Stoeger C, Terry MA. Endothelial keratoplasty 
1171 
using donor tissue not suitable for full-thickness penetrating keratoplasty. Cornea. 
1172 
2007;26(5):515-9. 
1173 
7. Chang SD, Pecego JG, Zadnik K, Danneffel MB, Mutti DO, Mannis MJ. Factors influencing 
1174 
graft clarity. Cornea. 1996;15:577-81. 
1175 
8.  Abbott RL, Forster RK. Determinants of graft clarity in penetrating kerotoplasty. Arch 
1176 
Ophthalmol. 1979;97(6):1071-5. 
1177 
9. Wagoner MD, Gonnah el S. Corneal graft survival after prolonged storage in Optisol-GS. 
1178 
Cornea. 2005;24(8):976-9. 
1179 
10. Doganay S, Hepsen IF, Yologlu S, Demirtas H. Effect of the preservation-to-surgery interval 
1180 
on corneal allograft survival in low-risk patients. Ophthalmic Surg Lasers Imaging. 
1181 
2007;38(6):457-61. 
1182 
11. Sugar A, Gal RL, Beck W, Ruedy KJ, Blanton CL, Feder RS, et al. Baseline donor  
1183 
characteristics in the Cornea Donor Study. Cornea. 2005;24(4):389-96. 
1184 
12. Guttman C. Donor Death-Surgery Interval May Affect DSAEK Outcomes. Ophthamology 
1185 
Times. April 15, 2009. 
1186 
13. Price MO, Price FW, Jr. Endothelial cell loss after descemet stripping with endothelial 
1187 
keratoplasty influencing factors and 2-year trend. Ophthalmology. 2008;115(5):857-65. 
1188 
14.  Chen ES, Terry MA, Shamie N, Hoar KL, Friend DJ. Precut Tissue in Descemet's Stripping  
1189 
     Automated Endothelial Keratoplasty Donor Characteristics and Early Postoperative 
1190 
       Complications. Ophthalmology. 2008. 
1191 
15. Terry MA, Chen ES, Shamie N, Hoar KL, Friend DJ. Endothelial cell loss after Descemet's 
1192 
stripping endothelial keratoplasty in a large prospective series. Ophthalmology. 
1193 
2008;115(3):488-96 e3. 
1194 
16. Terry MA. Precut tissue for descemet stripping automated endothelial keratoplasty: 
1195 
complications are from technique, not tissue. Cornea. 2008;27(6):627-9. 
1196 
17. Terry MA, Shamie N, Straiko MD, Friend DF. Endothelial keratoplasty:  The relationship 
1197 
between donor tissue storage time and donor endothelial survival. Ophthalmology.  
1198 
2011;118:36-40. 
1199 
18.  Louttit MD, Kopplin LJ, Igo, Jr RP, Fondran JR, Tagliaferri A, Bardenstein D, Aldave AJ, 
1200 
Croasdale CR, Price M, Rosenwasser GO, Lass JH, Iyengar SK:  A multi-center study to map 
1201 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
32 
genes for Fuchs’ Endothelial Corneal Dystrophy:  Baseline Characteristics and Heritability.  
1202 
Cornea In press 
 
1203 
19.  Eye Bank Association of America. Medical Standards. Washington, DC; 2009  
1204 
20.  Eye Bank Association of America. Procedures Manual. Washington, DC; 2009  
1205 
21. Collaborative Corneal Transplantation Studies Research Group. The collaborative corneal 
1206 
transplantation studies (CCTS). Effectiveness of histocompatibility matching in high-risk 
1207 
corneal transplantation. The Collaborative Corneal Transplantation Studies Research Group. 
1208 
Arch Ophthalmol. 1992;110(10):1392-403. 
1209 
22. Maguire MG, Stark WJ, Gottsch JD, Stulting RD, Sugar A, Fink NE, et al. Risk factors for 
1210 
corneal graft failure and rejection in the collaborative corneal transplantation studies. 
1211 
Collaborative Corneal Transplantation Studies Research Group. Ophthalmology. 
1212 
1994;101(9):1536-47. 
1213 
23.  Jordan C, Price M, et al.  Graft rejection episodes after Descemet stripping with endothelial   
1214 
keratoplasty: part one: clinical signs and symptoms. Br J Ophthalmol 2009: 387-390. 
1215 
 
1216 
 
1217 
 
1218 
1219 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
33 
APPENDIX A 
1220 
 
1221 
PROTOCOL AMENDMENT #1 
1222 
 
1223 
 
1224 
EXTENSION OF FOLLOW-UP TO COMMON END DATE 
1225 
 
1226 
July 1, 2015 
1227 
1228 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
34 
1. Overview:  Extension of follow-up after 3 years.  
1229 
 
Participants who re-consent to additional follow-up will continue annual protocol visits 
1230 
past the 3-year time point, until at least 2017.  The same data will be captured from annual visits 
1231 
as well as interim unscheduled visits as was done previously. The same study specific tests – 
1232 
pachymetry and specular/confocal microscopy - will continue to be performed at the annual 
1233 
visits. Visits will continue to be captured until approximately the 2nd quarter of 2017, therefore 
1234 
all participants will have a last possible common endpoint rather than exiting individually when 
1235 
they reach their 3 year post-operative visit.  
1236 
Rationale: Continued follow-up of CPTS participants provides an excellent opportunity to 
1237 
gain more information about many factors affecting DSAEK outcomes, including donor age 
1238 
and preservation time. This additional longitudinal data will provide important information 
1239 
on longer term DSAEK outcomes that are not necessarily impacted by preservation time, 
1240 
although we will continue to assess that variable as well.   
1241 
 
1242 
 
1243 
2. Eligibility and Informed Consent 
1244 
a. Eligibility 
1245 
All active study participants will be eligible for extended follow-up to a common end date, until 
1246 
at least 2017.  The exact end date will be determined by the Operations Committee based on 
1247 
funding and data analysis requirements.      
1248 
b. Informed Consent 
1249 
Active study participants will be asked to sign a new informed consent form or addendum prior 
1250 
to the post-3 year visits or as soon thereafter as feasible.  The new informed consent form (or 
1251 
addendum) may be signed during the next scheduled visit (either Protocol Visit or Unspecified 
1252 
Visit) or by mail if the governing IRB approves that process.   Until the new informed consent 
1253 
form or addendum is signed, the study participant will not be examined post-3 year for study 
1254 
purposes.  If the new informed consent form or addendum is not signed, follow-up for that 
1255 
participant will end upon completion of the 36-month visit. 
1256 
3. Follow-Up Visits 
1257 
 
1258 
a. Visit Schedule and Windows 
1259 
The post-3 year visit schedule will vary by the participant’s enrollment date.  Some participants 
1260 
will be eligible for Year 4 and Year 5 visits if the windows below fall within the extended 
1261 
follow-up period.  
1262 
 
1263 
Additional protocol-specified follow-up visits (and visit windows) for the first eye, will be as 
1264 
follows: 
1265 
x 48 months (44 - 52 months) 
1266 
x 60 months (56 – 64 months) 
1267 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
35 
Additional visits may be performed more often as needed.  A data form will be completed for 
1268 
each protocol visit and any non-protocol visits where the intent of the visit was to examine the 
1269 
study eye by a study investigator. Additional non-protocol visits by non-study investigators may 
1270 
also be uploaded by the clinical site to the study website, as was done during the initial 3-year 
1271 
follow-up phase. For example, if graft failure is determined and a regraft is required on a non-
1272 
protocol visit, the appropriate follow-up visit form and graft failure form should be completed if 
1273 
and when this occurs. 
1274 
If the second eye of an active participant was enrolled, a modified visit schedule will be 
1275 
allowed to minimize return visits for participants that had bilateral EK as part of the study. For 
1276 
example, sites will be allowed to follow standard of care practices to avoid unnecessary visits 
1277 
linked to targeting each eye within its respective windows as listed above.  Sites are encouraged 
1278 
to schedule both eyes within their respective windows when the windows overlap, but at 
1279 
minimum at least one eye must be within its target visit window at each visit.  
1280 
 
1281 
b. Testing Procedures 
1282 
The Table below shows the key elements of data collection at each study visit.  Additional 
1283 
visits may occur as needed for the usual care of the participant. 
1284 
 
1285 
 
1286 
 
1287 
 
1288 
 
1289 
 
1290 
 
1291 
 
1292 
 
1293 
 
1294 
 
1295 
 
1296 
c. Detailed Testing Procedures 
1297 
Procedures for testing at each follow-up visit are identical as listed in Chapter 4. 
1298 
 
1299 
d. Adverse Events 
1300 
Adverse event reporting remains identical to Chapter 5.  
1301 
 
1302 
e. Other Considerations in Follow-up 
1303 
All other retention and follow-up procedures including central contract from the 
1304 
Coordinating Center will continue as in the original study and as outlined in Chapter 6. 
1305 
 
48 
Months 
60 
Months 
Parameters 
 
 
Medication History 
X 
X 
Slit lamp examination 
X 
X 
Intraocular pressure 
X 
X 
Ultrasonic pachymetry 
X 
X 
Endothelial imaging 
X 
X 
Post-op complications and other 
untoward events  
X 
X 

Cornea Preservation Time Study; Version 4.0; July 1, 2015; NEI Grants:1U10EY020798-01A1 (J. Lass PI) and 
1U10EY020797-01 A1 (A. Edwards PI) 
 
36 
 
1306 
4. Statistical Analyses 
1307 
The statistical methods for all 3 study objectives completed up to the 3 year primary endpoint 
1308 
will be extended to the 4 and 5 year endpoints.  Additional analyses on the impact of missing 
1309 
data will be evaluated, including comparison of baseline characteristics for those who agreed 
1310 
versus declined to consent to continue.  
1311 
 
1312 
 
1313 
 
1314 
 
1315 
 
1316 
 
1317 
 
1318 
 
1319 
 
1320 
 
1321 

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϭŽĨϭϳ

 
ϭ
 
Ϯ
CPTS Statistical Analysis Plan 
ϯ
 
ϰ
Version: 4.0 
ϱ
Date: 6-21-16 
ϲ
In sync with Protocol Version 4.0 
ϳ
 
ϴ
 
 
ϵ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϮŽĨϭϳ

 
ϭϬ
Version History  
ϭϭ
 
ϭϮ
Version 
Number 
Author 
Approver 
Effective 
Date 
Revision Description 
1.0 
Allison 
Ayala 
Craig Kollman 
9-4-14 
Revisions prior to 9-4-14 were not 
versioned according to new JCHR 
standards.   Changes were clarifications 
from DSMC meetings and were 
documented in DSMC meeting minutes. 
2.0 
Allison 
Ayala  
Craig Kollman 
8-12-15 
Added analyses related to extension of 
CPTS to capture 4 and 5 year visits, which 
was incorporated into protocol version 
4.0.   
3.0 
Allison 
Ayala 
Craig Kollman 
1-15-16 
The DSMC, OC, and EC approved the 
clarification of how to handle cases that 
were cloudy at the 3 year visit.   
Details of the original discussion are in 
the November 3, 2015 DSMC minutes 
and details of the discussion/proposal that 
followed (and final decision) are in the 
following folder: Analysis Notes to 
Save\3 year failure cutoff discussions 
4.0  
Allison 
Ayala 
Craig Kollman 
6-21-16 
1. Clarify that “surgical trauma” (like 
blunt and penetrating trauma) is 
also a severe event, if leading to 
failure, that is unrelated to PT and 
should be censored prior to the 
event.  
2. Clarify that out of window 3 year 
visits can be used up to 44 months 
for determining 3 year failures. 
Decision/discussion saved in the 
following folder:  Analysis Notes 
to Save\3 year visit window  
 
 
 
ϭϯ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϯŽĨϭϳ

CPTS Statistical Analysis Plan 
ϭϰ
   
ϭϱ
1.0 Study Objectives  
ϭϲ
The objectives of the “Effect of Corneal Preservation Time on Long-Term Graft Success” (CPTS) 
ϭϳ
study are: 
ϭϴ
 
ϭϵ
Objective 1 (Primary Objective): To determine if the 3-year graft failure rate following EK 
ϮϬ
performed with donor corneas with a preservation time of 8 to14 days is non-inferior to the 
Ϯϭ
failure rate when donor corneas with a preservation time of 7 or fewer days are used. 
ϮϮ
 
Ϯϯ
Objective 2: To determine if the central corneal endothelial cell density 3-years after EK is 
Ϯϰ
related to preservation time. 
Ϯϱ
 
Ϯϲ
Objective 3:  To evaluate the effect of donor, operative and postoperative factors on graft 
Ϯϳ
failure and endothelial cell density three years following EK. 
Ϯϴ
 
Ϯϵ
The purpose of this document is to describe the analysis plan for these 3 objectives, as well as some   
ϯϬ
pre-planned secondary analyses.  There may be additional secondary analyses performed that are not 
ϯϭ
described in this document. 
ϯϮ
 
ϯϯ
2.0 Sample Size 
ϯϰ
The sample size of 1330 is calculated based on a non-inferiority (a single one-sided test) design with 
ϯϱ
the goal to determine that the graft failure rate of the recipients of donor tissue transplanted 8 to 14 
ϯϲ
days after preservation is not worse than the graft failure rate of recipients of donor tissue transplanted 
ϯϳ
< 7 days after preservation. 
ϯϴ
 
ϯϵ
Non-Inferiority 
Limit 
Power = 90% 
Failure Rate 
12% 
10% 
8%a 
6% 
4% 
10% 
362 
310 
 
 
 
8% 
566 
482 
394 
 
 
6% 
1006 
858 
702 
538 
 
4 % 
2262 
1928 
1576 
1208 
824 
2% 
9044 
7708 
6304 
4832 3290 
Note: Numbers in table are total sample size for both treatment groups combined 
ϰϬ
(crossover and lost to follow up are not accounted for). Half would be randomized to 
ϰϭ
each group. 
ϰϮ
a CDS data: 3-year failure rate 
ϰϯ
 
ϰϰ
In the Cornea Donor Study (CDS), the 3-year failure rate was 8%.  Clinical expectations suggest that 
ϰϱ
the graft failure rate from the EK procedure will be smaller than the failure rate from the PKP 
ϰϲ
procedure that was used in CDS; therefore, a 3-year failure rate of 6% has been assumed.  Based on 
ϰϳ
equal allocation of recipients to each group and type I error of 5%, a sample size of 1208 will provide 
ϰϴ
90% power for a non-inferiority limit of 4%.  Based on information from the CDS, approximately 
ϰϵ
10% of subjects will have incomplete follow up (due to death, withdrawal or lost to follow up) by the 
ϱϬ
end of year 3.  Increasing the calculated sample size by this amount gives a total of 1330 subjects 
ϱϭ
(665 per group). 
ϱϮ
• The inclusion of participants with two study eyes, one in each treatment group, will tend to 
ϱϯ
reduce the variance and as a result increase statistical power. To be conservative, this has not 
ϱϰ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϰŽĨϭϳ

been accounted for in the sample size estimation, since the correlation of outcome with two 
ϱϱ
eyes is not known. 
ϱϲ
 
ϱϳ
 
ϱϴ
3.0 Graft Failure (Primary Outcome)   
ϱϵ
 
ϲϬ
3.1 Primary Analysis of 3 Year Graft Failure  - (Analysis plan for objective 1) 
ϲϭ
 
ϲϮ
3.1.1 Formal Statistical Hypothesis 
ϲϯ
The primary objective of the study is to determine if the 3-year graft failure rate 
ϲϰ
following EK performed with donor corneas with a preservation time of 8 to14 days 
ϲϱ
is non-inferior to the failure rate when donor corneas with a preservation time of 7 or 
ϲϲ
fewer days are used.  In terms of formal statistical hypothesis testing, the null and 
ϲϳ
alternative hypotheses are: 
ϲϴ
 
ϲϵ
Ho: p8-14 – p0-7 > 4%    
ϳϬ
Ha: p8-14 – p0-7 < 4%    
ϳϭ
 
ϳϮ
where p8-14 and p0-7  is the probability of graft failure by 3 years in the 8-14 day group 
ϳϯ
and the <=7 day group, respectively. 
ϳϰ
 
ϳϱ
3.1.2 Analysis Cohort 
ϳϲ
Participant study eyes that did not have surgery, received a non-study donor, had an 
ϳϳ
AC IOL implanted during surgery, or experienced a suprachoroidal hemorrhage will 
ϳϴ
be excluded from the primary analysis. It is highly unlikely these events could be 
ϳϵ
related to preservation time, thus inclusion of them could actually bias towards 
ϴϬ
concluding non-inferiority. Therefore, the primary analysis will deviate from the 
ϴϭ
principle of intent-to-treat, but this approach is conservative for a non-inferiority 
ϴϮ
analysis.   
ϴϯ
 
ϴϰ
3.1.2.1 Censoring of Data for Occurrence of a Severe Event Unrelated to 
ϴϱ
Preservation Time 
ϴϲ
In order to minimize bias towards concluding non-inferiority, severe events 
ϴϳ
not expected to be related to preservation time will be censored at the last 
ϴϴ
examination prior to the occurrence of the event that severely impacts the study 
ϴϵ
eye, if the eye was not on the path to failure at the last visit prior to the 
ϵϬ
occurrence of the severe unrelated event (see 3.1.5 for the ‘rules’). These 
ϵϭ
events include: 
ϵϮ
 Enucleation  (eye will be dropped) 
ϵϯ
 Phthisis (eye will be dropped) 
ϵϰ
 Failure due to surgical (unrelated to the initial DSEK), blunt or 
ϵϱ
penetrating trauma (eye will be followed until failure criteria met) 
ϵϲ
 
ϵϳ
3.1.2.2 Analyzing Preservation Time Group Crossovers As-Treated 
ϵϴ
If a study eye receives a study donor cornea in the wrong preservation time 
ϵϵ
group, it will be analyzed as treated, as this will minimize bias towards 
ϭϬϬ
concluding non-inferiority.   A secondary intent-to-treat analysis will also be 
ϭϬϭ
completed if this occurs. 
ϭϬϮ
 
ϭϬϯ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϱŽĨϭϳ

 
ϭϬϰ
3.1.3 Definition of Graft Failure (per protocol,  section 4.7): 
ϭϬϱ
Graft failure will be assessed and defined as the occurrence of one of the following: 
ϭϬϲ
• Cornea which requires regrafting for any reason 
ϭϬϳ
• Cornea which remains cloudy without clearing, according to the following:    
ϭϬϴ
(1)  cloudy cornea on the first postoperative day which does not clear 
ϭϬϵ
within 8 weeks    
ϭϭϬ
 
 
OR  
ϭϭϭ
(2) cloudy cornea which was initially clear postoperatively but becomes 
ϭϭϮ
and remains cloudy for 3 months without clearing.    
ϭϭϯ
Note: graft failure is based on cloudy recipient stroma. Any reference to 
ϭϭϰ
cloudy cornea when defining graft failure is with regard to the recipient 
ϭϭϱ
stromal clarity.  
ϭϭϲ
For eyes meeting the definition of graft failure above, the principal cause of graft 
ϭϭϳ
failure will be classified as one of the following:  
ϭϭϴ
• Early failure (cloudy or equivocal recipient cornea on the first postoperative 
ϭϭϵ
day which does not clear or requires a regraft within 8 weeks), associated 
ϭϮϬ
with 
surgical 
complications, 
including 
immediate 
peri-operative 
ϭϮϭ
complications such as acute angle closure noted on the 1 day post-operative 
ϭϮϮ
visit   
ϭϮϯ
• Primary donor failure (cloudy or equivocal recipient cornea on the first 
ϭϮϰ
postoperative day which does not clear or requires a regraft within 8 weeks),  
ϭϮϱ
in the absence of surgical complications   
ϭϮϲ
• Graft rejection (defined as a clouded recipient central stroma following an 
ϭϮϳ
allograft reaction);  
ϭϮϴ
• Non-rejection graft failure (defined as a graft that initially had a clear central 
ϭϮϵ
recipient stroma and becomes cloudy due to causes other than an immune 
ϭϯϬ
event.  These include:  surface failure,  infection, glaucoma/hypotony, 
ϭϯϭ
endothelial decompensation,  interface irregularity or opacity, pre-existing 
ϭϯϮ
stromal scarring, blunt or penetrating trauma, and other causes);  
ϭϯϯ
• Refractive/visual graft failure (defined as a graft that requires regrafting due 
ϭϯϰ
to inadequate vision while the recipient central stroma remains clear).  
ϭϯϱ
 
ϭϯϲ
3.1.4 Definition of Graft Failure Date 
ϭϯϳ
A cornea that is “on the path to failure” means the cornea has met criteria to initiate 
ϭϯϴ
the path to failure and it has not been removed from that path, defined as follows: 
ϭϯϵ
1. Definition of how to initiate “on the path to failure”  : 
ϭϰϬ
• Initiating At 1 Day Visit: A cornea may be classified as either cloudy or 
ϭϰϭ
equivocally cloudy to start a count of how many days “on the path to 
ϭϰϮ
failure”. 
ϭϰϯ
• Initiating After 1 Day Visit:   A cornea must be initially classified as cloudy 
ϭϰϰ
to start a count of how many days “on the path to failure”. 
ϭϰϱ
2. A cornea classified as equivocal after the “on the path to failure” count begins is 
ϭϰϲ
still considered “on the path to failure.” 
ϭϰϳ
3. A cornea classified as clear after the “on the path to failure” count begins is no 
ϭϰϴ
longer “on the path to failure”; the count restarts the next time the cornea is 
ϭϰϵ
classified as cloudy. 
ϭϱϬ
   
 
ϭϱϭ
The date of graft failure is defined as follows   
ϭϱϮ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϲŽĨϭϳ

1. If “on the path to failure” (per 3.1.4 above) initiates at the 1 Day Visit:  
ϭϱϯ
a. If the cornea remains “on the path to failure” (per 3.1.4 above), is 
ϭϱϰ
classified cloudy at least once during those consecutive visits, and is 
ϭϱϱ
classified cloudy  at least 56 days after surgery date, then the cornea meets 
ϭϱϲ
the failure definition and the date of failure will be the date of the 1 day 
ϭϱϳ
visit.   (Note: this means it must be classified cloudy at least twice during 
ϭϱϴ
the path to failure, and at least one of those >= 56 days after surgery) 
ϭϱϵ
b. If the cornea remains “on the path to failure” (per 3.1.4 above) and a 
ϭϲϬ
regraft occurs within 56 days or after 56 days (but prior to being classified 
ϭϲϭ
cloudy after 56 days), the date of failure will equal the date of the 1 day 
ϭϲϮ
visit.   
ϭϲϯ
2. If “on the path to failure” (per 3.1.4 above) initiates after the 1 Day Visit:  
 
ϭϲϰ
a. If a cornea is classified as cloudy, remains “on the path to failure” (per 
ϭϲϱ
3.1.4 above), and is classified cloudy at least 90 days after the initial 
ϭϲϲ
cloudy classification, then the cornea meets the failure definition and the 
ϭϲϳ
date of failure will be the first date at which cornea is indicated as cloudy.  
ϭϲϴ
b. If a cornea is “on the path to failure” (per 3.1.4 above) for <90 days and a 
ϭϲϵ
regraft occurs, the date of failure will equal the first exam date where the 
ϭϳϬ
cornea is cloudy. 
ϭϳϭ
3. If a cornea is clear and then a regraft occurs, the date of failure will be equal to 
ϭϳϮ
the date of regraft.   
ϭϳϯ
 
 
ϭϳϰ
Examples 
 
ϭϳϱ
Example 1 
ϭϳϲ
equivocal (1 day)  
 Å failure date 
ϭϳϳ
equivocal    
ϭϳϴ
cloudy   (>56 days after surgery)  
ϭϳϵ
cloudy (need the second cloudy to confirm) 
ϭϴϬ
 
ϭϴϭ
 
ϭϴϮ
Example 2 
ϭϴϯ
equivocal (1 day)  
 Å failure date 
ϭϴϰ
equivocal    
ϭϴϱ
regraft 
ϭϴϲ
 
ϭϴϳ
Example 3 
ϭϴϴ
clear  
ϭϴϵ
equivocal 
ϭϵϬ
cloudy*     
 
Å failure date  
ϭϵϭ
equivocal 
ϭϵϮ
cloudy   (>90 days after *) 
ϭϵϯ
 
ϭϵϰ
 Example 4 
ϭϵϱ
clear   
ϭϵϲ
cloudy 
ϭϵϳ
clear   
ϭϵϴ
cloudy*  
 
Å failure date  
ϭϵϵ
cloudy   (<90 days after *) 
ϮϬϬ
regraft 
ϮϬϭ
 
ϮϬϮ
Example 5 
ϮϬϯ
clear   
ϮϬϰ
clear    
ϮϬϱ
regraft  
 
Å failure date 
ϮϬϲ
 
ϮϬϳ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϳŽĨϭϳ

 
ϮϬϴ
 
ϮϬϵ
3.1.5 Censoring and Non-Protocol Graft Failures 
ϮϭϬ
3.1.5.1 Eyes with Incomplete Follow-Up 
Ϯϭϭ
In eyes that dropped prior to the 3 year visit without meeting criteria for graft 
ϮϭϮ
failure the following ‘rules’ will apply:  
Ϯϭϯ
1. A cornea that is “on the path to failure” (per 3.1.4 above) at the last 
Ϯϭϰ
completed visit AND was cloudy at least once during the “on the path 
Ϯϭϱ
to failure” period  will be flagged for ‘non-protocol’ graft failure 
Ϯϭϲ
review by the Executive Committee.  If confirmed as a non-protocol 
Ϯϭϳ
graft failure, the failure date will be determined as it is in 3.1.4 above.   
Ϯϭϴ
2. All others will NOT be considered a graft failure and the data will be 
Ϯϭϵ
censored at the last completed visit  
ϮϮϬ
 
 
ϮϮϭ
3.1.5.2 Eyes with 3 Year Visit Not Meeting Failure Criteria  
ϮϮϮ
In eyes that complete the 3 year visit without meeting criteria for graft failure 
ϮϮϯ
the following ‘rules’ will apply:   
ϮϮϰ
1. If the cornea is “on the path to failure” (per 3.1.4 above) at the 3 year 
ϮϮϱ
visit, data beyond the 3 year visit up to 42 months will be used (if 
ϮϮϲ
available) to determine whether the cornea will be considered a failure 
ϮϮϳ
up to 3 years.  Data beyond 42 months will not be used for this 
ϮϮϴ
assessment.  
ϮϮϵ
 If the cornea clears at a subsequent follow-up visit (within 42 
ϮϯϬ
months) prior to meeting confirmation of failure criteria, the 
Ϯϯϭ
cornea will not be classified as a graft failure and data will be 
ϮϯϮ
censored at the 3 year visit. 
Ϯϯϯ
 If additional follow-up data (within 42 months) confirm a 
Ϯϯϰ
failure (via regraft or 90 days confirmed cloudy), then the 
Ϯϯϱ
cornea will be classified as a graft failure up to 3 years and 
Ϯϯϲ
date of failure will be determined per 3.1.4 above. 
Ϯϯϳ
 If data beyond the 3 year visit are not available, OR the cornea 
Ϯϯϴ
remains “on the path to failure” beyond the 3 year visit but the 
Ϯϯϵ
follow-up data that are available within 42 months still do not 
ϮϰϬ
confirm failure,  the case will be flagged for ‘non-protocol’ 
Ϯϰϭ
graft failure review by the Executive Committee.  (Note all 
ϮϰϮ
of these cases will have been cloudy at least once during the 
Ϯϰϯ
“on the path to failure” period.)  If confirmed as a non-protocol 
Ϯϰϰ
graft failure, the failure date will be determined per 3.1.4 
Ϯϰϱ
above.   
Ϯϰϲ
2. If the cornea is not “on the path to failure” (clear or equivocal following 
Ϯϰϳ
clear) at the 3 year visit, it will NOT be considered a graft failure and 
Ϯϰϴ
the data will be censored at the 3 year visit   
Ϯϰϵ
 
ϮϱϬ
3.1.5.3 Severe Events Unrelated to Preservation Time  
Ϯϱϭ
In eyes that   met failure due to a severe event unrelated to preservation time 
ϮϱϮ
(listed in section 3.1.2.1), the following ‘rules’ will apply:  
Ϯϱϯ
1. A cornea that is “on the path to failure” (per 3.1.4 above) at the last 
Ϯϱϰ
completed visit  prior to  the severe event leading to  failure AND was 
Ϯϱϱ
cloudy at least once during that “on the path to failure” period prior to 
Ϯϱϲ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϴŽĨϭϳ

the event  will be flagged for ‘non-protocol’ graft failure review by 
Ϯϱϳ
the Executive Committee.  If confirmed as a non-protocol graft 
Ϯϱϴ
failure, the failure date will be determined as it is in 3.1.4 above.   
Ϯϱϵ
2. All others will NOT be considered a graft failure and the data will be 
ϮϲϬ
censored at the last completed visit  prior to the severe event leading 
Ϯϲϭ
to failure. 
ϮϲϮ
Examples 
Ϯϲϯ
Example 1 
Ϯϲϰ
clear  
Ϯϲϱ
clear  
Ϯϲϲ
cloudy     
Å ‘non-protocol’ failure date 
Ϯϲϳ
equivocal   
Ϯϲϴ
lost to followup 
Ϯϲϵ
 
ϮϳϬ
Example 2 
Ϯϳϭ
clear  
ϮϳϮ
clear  
Ϯϳϯ
equivocal 
Ϯϳϰ
equivocal 
   Å censored date  
Ϯϳϱ
lost to followup 
Ϯϳϲ
  
Ϯϳϳ
Example 3 
Ϯϳϴ
clear  
Ϯϳϵ
clear  
ϮϴϬ
cloudy     
Å ‘non-protocol’ failure date  
Ϯϴϭ
blunt trauma 
ϮϴϮ
cloudy 
Ϯϴϯ
regraft 
Ϯϴϰ
 
Ϯϴϱ
Example 4 
Ϯϴϲ
clear   
Ϯϴϳ
cloudy 
Ϯϴϴ
clear   
 
Å censored date 
Ϯϴϵ
blunt trauma 
ϮϵϬ
cloudy 
Ϯϵϭ
regraft 
ϮϵϮ
 
Ϯϵϯ
Example 5 
Ϯϵϰ
equivocal (1 day)  
  
Ϯϵϱ
equivocal 
 
Å censored date 
Ϯϵϲ
lost to followup 
Ϯϵϳ
 
Ϯϵϴ
 
Ϯϵϵ
Example 6 
ϯϬϬ
equivocal (1 day) Å ‘non-protocol’ failure date 
ϯϬϭ
cloudy 
ϯϬϮ
lost to followup 
ϯϬϯ
 
ϯϬϰ
3.1.6 Clarifications Regarding 3 Year Visit Windows 
ϯϬϱ
The visit that the site designates as the “3 year visit” is the one that drives whether a 3 
ϯϬϲ
year failure has occurred.  If the eye is dropped without meeting failure and prior to 
ϯϬϳ
completion of the 3 year visit, the rules in 3.1.5.1 apply.  If failure definition is met 
ϯϬϴ
prior to a 3 year visit, then this is counted as a 3 year failure according to 3.1.4.   If 
ϯϬϵ
failure is not met prior to a 3 year visit, the rules in 3.1.5.2 are followed.  The following 
ϯϭϬ
clarifications regarding visit windows will apply to these rules: 
ϯϭϭ
 
ϯϭϮ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϵŽĨϭϳ

• The window for the 3 year visit, per protocol, is 35-42 months.  The 3 year 
ϯϭϯ
visit will be permitted to occur late out of window, up to 44 months.   For a 3 
ϯϭϰ
year visit occurring late out of window (between 42-44 months) the same rules 
ϯϭϱ
above apply relative to this designated 3 year visit. 
ϯϭϲ
• If there is no designated 3 year visit completed, data (i.e. unspecified visits) up 
ϯϭϳ
to 44 months can be used to determine if failure met. The same rules above 
ϯϭϴ
will apply.     
ϯϭϵ
• NOTE: This means that data beyond 42 months (up to 44 months) CAN be 
ϯϮϬ
used to determine 3 year failure status if and only if the visits occur prior to or 
ϯϮϭ
including a designated 3 year visit late out of window, OR in the absence of a 
ϯϮϮ
designated 3 year visit.       
ϯϮϯ
 
ϯϮϰ
 
ϯϮϱ
 
ϯϮϲ
3.1.7 Unadjusted Analysis 
ϯϮϳ
Three year Kaplan-Meier graft failure estimates with 95% confidence intervals 
ϯϮϴ
(variance estimated using the Greenwood method) will be calculated separately for 
ϯϮϵ
the two treatment groups (7 days and 8 to 14 days from preservation to surgery). A 
ϯϯϬ
one-sided 95% confidence interval will be constructed for the difference in 3 year graft 
ϯϯϭ
failure rates between the two groups. The bootstrap re-sampling technique will be used 
ϯϯϮ
to account for potentially correlated data from donors who donated both corneas in 
ϯϯϯ
this study and potentially correlated data from 2 study eyes of the same study 
ϯϯϰ
participant. The technique will sample with replacement from the observed dataset.  
ϯϯϱ
For each bootstrap sample the effect of preservation time will be estimated using the 
ϯϯϲ
same method as in the primary analysis.  Confidence intervals will be calculated using 
ϯϯϳ
the bias-corrected and accelerated method.  The number of bootstraps will be 100,000. 
ϯϯϴ
 
 
ϯϯϵ
The two treatment groups will be declared equivalent if the one-sided 95% confidence 
ϯϰϬ
interval for the difference in proportions excludes the pre-defined non-inferiority limit 
ϯϰϭ
of 4%. 
ϯϰϮ
 
ϯϰϯ
3.1.8 Adjusted Analysis 
ϯϰϰ
Multivariate analysis will be performed using Cox proportional hazards regression 
ϯϰϱ
model.  The primary multivariate model will include the corneal diagnosis regardless 
ϯϰϲ
of statistical significance, in addition to time from preservation to surgery (treated as 
ϯϰϳ
a binary variable).   
ϯϰϴ
 
ϯϰϵ
In additional models, potential confounders including recipient and donor age, 
ϯϱϬ
recipient and donor race, presence of glaucoma, presence of corneal vessels, history 
ϯϱϭ
of smoking, and certain aspects of the retrieval and processing of the donor tissue 
ϯϱϮ
(including multiple types of storage media, if more than one is used in preservation of 
ϯϱϯ
the corneal tissues, observations during or after cutting, time from cut to surgery, and 
ϯϱϰ
donor rim culture results) will be screened by assessing the change in the preservation 
ϯϱϱ
time effect when the potential confounder is controlled for in the Cox model. 
ϯϱϲ
Univariate models will be evaluated first, and factors from those models with a p value 
ϯϱϳ
<0.10 will then be evaluated in a multivariate model.  A final model will be 
ϯϱϴ
constructed consisting of factors with a p value <0.01 following a backwards selection 
ϯϱϵ
process.    
ϯϲϬ
 
ϯϲϭ
3.1.9 Surgeon Effect 
ϯϲϮ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϭϬŽĨϭϳ

Random surgeon effects will be tested using a generalized linear model via the SAS 
ϯϲϯ
GLIMMIX procedure.  This marginal model produces a robust standard error (RSE) 
ϯϲϰ
by use of a sandwich estimator, which corrects for correlated data. 
ϯϲϱ
 
ϯϲϲ
3.1.10 Analysis of Potential Interaction  
ϯϲϳ
Potential effect modifiers of donor tissue preservation time such as recipient age or 
ϯϲϴ
corneal diagnosis will be screened by including first-order interaction terms.  
ϯϲϵ
Variables that exhibit modification of the donor tissue preservation time effect with 
ϯϳϬ
an associated P value < 0.10 will be added to the model, and the final model will be 
ϯϳϭ
constructed consisting of terms with a p value <0.01 following a backwards selection 
ϯϳϮ
process. 
ϯϳϯ
 
ϯϳϰ
 
ϯϳϱ
 
ϯϳϲ
3.2 Secondary Analyses of 3 Year Graft Failure 
ϯϳϳ
 
ϯϳϴ
3.2.1 Preservation Time 
ϯϳϵ
The time from preservation to surgery is treated as a binary variable in the primary 
ϯϴϬ
analysis (see above section).  Secondary analyses will look at the time from 
ϯϴϭ
preservation to surgery as a categorical variable with multiple levels and as a 
ϯϴϮ
continuous variable: 
ϯϴϯ
• Kaplan-Meier estimates of graft failure with 95% confidence interval will be 
ϯϴϰ
calculated for each of the following groups: 0 – 4, 5 – 7, 8 – 11, and 12 – 14 days 
ϯϴϱ
from preservation to surgery (counting partial days as whole days).   
ϯϴϲ
• A Cox proportional hazards model will be constructed treating the time from 
ϯϴϳ
preservation to surgery as a continuous variable (using time of day to calculate 
ϯϴϴ
hours from preservation to surgery).  Polynomial terms will be added to assess any 
ϯϴϵ
curvilinear, J, or U shaped relationship between time from preservation to surgery 
ϯϵϬ
and graft failure.  If no significant departure from a linear relation is detected, a 
ϯϵϭ
one sided 95% confidence interval will be computed for the hazard ratio per day 
ϯϵϮ
of time from preservation to surgery. 
ϯϵϯ
 The proportional hazards assumptions will be tested through the use of time-
ϯϵϰ
dependent variables with a logarithm transformation of time.  If this 
ϯϵϱ
assumption is violated then hazard ratios will be presented separately for 
ϯϵϲ
different periods following transplant. 
ϯϵϳ
 
ϯϵϴ
3.2.2 Predictive Factors – (Analysis plan for objective 3, graft failure outcome) 
ϯϵϵ
The association of factors potentially related to graft failure will be evaluated in 
ϰϬϬ
univariate and multivariate Cox models , adjusting for preservation time group 
ϰϬϭ
regardless of statistical significance. The proportional hazards assumptions will be 
ϰϬϮ
tested as described above. Univariate models will be evaluated first, and factors from 
ϰϬϯ
those models with a p value <0.10 will then be evaluated in a multivariate model.  A 
ϰϬϰ
final model will be constructed consisting of factors with a p value <0.01 following a 
ϰϬϱ
backwards selection process.      
ϰϬϲ
 
 
ϰϬϳ
Potential factors to evaluate include: 
ϰϬϴ
• Recipient factors 
ϰϬϵ
¾ 
preoperative diagnosis 
ϰϭϬ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϭϭŽĨϭϳ

¾ 
gender 
ϰϭϭ
¾ 
age 
ϰϭϮ
¾ 
race  
ϰϭϯ
¾ 
prior use of glaucoma medication 
ϰϭϰ
¾ 
prior glaucoma surgery (trabeculectomy, laser procedure) 
ϰϭϱ
¾ 
current smoker (at time of surgery) 
ϰϭϲ
¾ 
lens status (phakic, posterior chamber intraocular lens) 
ϰϭϳ
¾ 
Intraocular pressure (IOP)  
ϰϭϴ
 
ϰϭϵ
• Donor/graft factors 
ϰϮϬ
¾ 
eye bank determined screening ECD  
ϰϮϭ
¾ 
pre-operative CIARC determined ECD 
ϰϮϮ
¾ 
age 
ϰϮϯ
¾ 
gender 
ϰϮϰ
¾ 
race 
ϰϮϱ
¾ 
history of diabetes 
ϰϮϲ
¾ 
cause of death 
ϰϮϳ
¾ 
type of storage medium 
ϰϮϴ
¾ 
death to preservation time 
ϰϮϵ
¾ 
surgeon cut vs eye bank cut 
ϰϯϬ
¾ 
postcut thickness 
ϰϯϭ
¾ 
cut to surgery time 
ϰϯϮ
¾ 
observations during or after cutting 
ϰϯϯ
 
ϰϯϰ
• Surgical factors  
ϰϯϱ
¾ 
Insertion method 
ϰϯϲ
¾ 
Incision location 
ϰϯϳ
¾ 
Incision site 
ϰϯϴ
¾ 
Graft size 
ϰϯϵ
 
ϰϰϬ
• Postoperative factors  
ϰϰϭ
¾ 
Dislocation 
ϰϰϮ
¾ 
Rebubbling 
ϰϰϯ
¾ 
IOP  
ϰϰϰ
¾ 
Graft rejection 
ϰϰϱ
¾ 
Corneal thickness 
ϰϰϲ
 
ϰϰϳ
 
ϰϰϴ
3.2.3 ECD as Time Dependent Predictor of Graft Failure 
ϰϰϵ
The relationship between endothelial graft failure (graft failure due to endothelial 
ϰϱϬ
decompensation) and ECD will be addressed paralleling the methods used in the CDS.  
ϰϱϭ
A Cox model will be fit with ECD as a time dependant covariate. This analysis will 
ϰϱϮ
be limited to subjects with at least one gradable follow up image.  The rate of change 
ϰϱϯ
will also be calculated as a time dependent variable defined as the least squares slope 
ϰϱϰ
over all previous measurements starting at 6 months (e.g., the rate of change at one 
ϰϱϱ
year would be the slope fit to the 6 month and 1 year ECD values).  Missing values 
ϰϱϲ
will be imputed by Rubin’s method.  If non-linear effects are detected, transformations 
ϰϱϳ
will be used.  For ease of interpretation, results will be presented as discrete categories 
ϰϱϴ
with cutpoints chosen to display the trends identified from the transformed model.  P-
ϰϱϵ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϭϮŽĨϭϳ

values will still be from a continuous analysis although data are displayed as 
ϰϲϬ
categories.  The proportional hazards assumptions will be tested as described above. 
ϰϲϭ
 
ϰϲϮ
To check whether results are sensitive to how missing data are handled, a second 
ϰϲϯ
model will be fit with a time dependent indicator for missing ECD. 
ϰϲϰ
 
ϰϲϱ
3.2.4 Secondary Outcome of Graft Rejection 
ϰϲϲ
Associations of baseline recipient and donor factors with the occurrence of a graft 
ϰϲϳ
rejection will be assessed in univariate and multivariate proportional hazards models. 
ϰϲϴ
Life-table analyses will be used to compute the probability of a first rejection event 
ϰϲϵ
within intervals defined by the study exam schedule.  Data will be censored at the time 
ϰϳϬ
of a non-rejection graft failure or at the last visit.   
ϰϳϭ
A Kaplan-Meier approach will be considered for evaluating probability of first graft 
ϰϳϮ
rejection, and time dependency of repeated rejection events will also be explored.   
ϰϳϯ
Determination of the timing of a separate episode graft rejection will include a 
ϰϳϰ
confirmation that the eye was off steroids at the visit where rejection was reported. 
ϰϳϱ
 
ϰϳϲ
4.0 Endothelial Cell Density (ECD)    
ϰϳϳ
 
ϰϳϴ
4.1  Primary Analysis of  3 Year ECD– (Analysis plan for objective 2) 
ϰϳϵ
4.1.1 Analysis Cohort 
ϰϴϬ
The primary analysis will include all study participants with a gradable 3-year image, 
ϰϴϭ
who have not experienced graft failure 3 years after transplantation.  
ϰϴϮ
 
ϰϴϯ
4.1.2 Outcome Measure  
ϰϴϰ
The primary outcome measure will be the ECD at 3 years, conditional on graft survival 
ϰϴϱ
at 3 years.   
ϰϴϲ
 
ϰϴϳ
4.1.3 Descriptive Statistics 
ϰϴϴ
• Summary statistics (mean ± SD and/or median/quartiles as appropriate to the 
ϰϴϵ
distribution) will be given for the ECD by the 2 treatment groups (7 and 8 to 14 
ϰϵϬ
days) and 4 treatment groups (0 – 4, 5 – 7, 8 – 11, and 12 – 14 days). 
ϰϵϭ
• Change from eye bank determined screening ECD will be summarized in a similar 
ϰϵϮ
manner. 
ϰϵϯ
• Boxplots of ECD and change from eye bank determined screening ECD will be 
ϰϵϰ
given for the 2 randomization groups.   
ϰϵϱ
• A scatter plot will be constructed of eye bank determined screening ECD vs. 3 
ϰϵϲ
year ECD with a symbol used to denote the two randomization groups. 
ϰϵϳ
 
ϰϵϴ
4.1.4 Analysis   
ϰϵϵ
An ANOVA model with 3 year ECD as the dependent variable will be used to assess 
ϱϬϬ
the effect of preservation time.   
ϱϬϭ
• Confounding with regard to screening ECD is not expected to be an issue due to 
ϱϬϮ
anticipated balance via randomization.   Therefore, an ANCOVA model adjusting 
ϱϬϯ
for eye-bank-determined screening ECD will only be used if this measurement is 
ϱϬϰ
considered good enough to expect to have any impact on reducing variance.    
ϱϬϱ
o Although all screening images are being collected, CIARC is not grading 
ϱϬϲ
them other than a general quality assessment.   CIARC will grade a sample 
ϱϬϳ
of screening images within each eye bank.   If more than 75% of the graded 
ϱϬϴ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ

WĂŐĞϭϯŽĨϭϳ

ECDs are within 10% of the eye bank determined ECDs, the eye bank 
ϱϬϵ
determined ECDs will be included in the model.   
ϱϭϬ
• The time from preservation to surgery will be treated as a binary variable.  
ϱϭϭ
• If residual values from the models above are highly skewed then a transformation 
ϱϭϮ
(e.g., square root or logarithm) or non-parametric methods will be used instead. 
ϱϭϯ
• Random effects will be modeled to account for any correlated data from the same 
ϱϭϰ
donor and any correlated data from 2 study eyes of the same study participant. 
ϱϭϱ
• Additional ANCOVA models will also adjust for other recipient/donor risk 
ϱϭϲ
factors.  Univariate models will be evaluated first, and factors from those models 
ϱϭϳ
with a p value <0.10 will then be evaluated in a multivariate model.  A final model 
ϱϭϴ
will be constructed consisting of factors with a p value <0.01 following a 
ϱϭϵ
backwards selection process 
ϱϮϬ
• Random surgeon effects will also be explored using a mixed effects model.  
ϱϮϭ
• Sensitivity analysis will also be performed to check whether results change 
ϱϮϮ
meaningfully depending on how missing data are handled. The missing 3 year 
ϱϮϯ
ECD values for subjects with surviving grafts at 3 years will be imputed and 
ϱϮϰ
included in an analysis as described in the previous section. The data imputation 
ϱϮϱ
will be performed by using Rubin’s method of multiple imputation. 
ϱϮϲ
 
ϱϮϳ
4.2 Secondary Analyses of 3 Year ECD 
ϱϮϴ
  
ϱϮϵ
4.2.1 Preservation Time 
ϱϯϬ
The time from preservation to surgery is treated as a binary variable in the primary 
ϱϯϭ
analysis of ECD (see above section).  The analyses described above will be repeated 
ϱϯϮ
with time from preservation to surgery treated as continuous (using time of day to 
ϱϯϯ
calculate hours from preservation to surgery) or multi-category variable (0 – 4, 5 – 7, 
ϱϯϰ
8 – 11, and 12 – 14 days from preservation to surgery) in separate models. 
ϱϯϱ
 
ϱϯϲ
4.2.2 Longitudinal analysis 
ϱϯϳ
This analysis also will be limited to subjects with a surviving graft at 3 years. A 
ϱϯϴ
repeated measures least squares regression model will be fit using all available images 
ϱϯϵ
at baseline, 6 months, 1, 2, and 3 years. This analysis will be performed with and 
ϱϰϬ
without imputation of missing data. Rubin’s method of data imputation will be used 
ϱϰϭ
to impute the ECD values for all missing time points. The time from preservation to 
ϱϰϮ
surgery will be modeled as both continuous and categorical as described above. If 
ϱϰϯ
residual values have a skewed distribution then transformation (e.g., square root or 
ϱϰϰ
logarithm) or non-parametric analysis will be used. 
ϱϰϱ
 
ϱϰϲ
4.2.3 Predictive factors – (Analysis plan for objective 3, ECD outcome) 
ϱϰϳ
This analysis also will be limited to subjects with a surviving graft at 3 years.    The 
ϱϰϴ
association of factors potentially related to 3 year ECD will be evaluated in 
ϱϰϵ
univariate and multivariate ANCOVA models , adjusting for preservation time group 
ϱϱϬ
regardless of statistical significance, and the reading center grading of pre-operative 
ϱϱϭ
ECD (imaged post-cut if the eye bank was performing the cutting and imaged just 
ϱϱϮ
prior to shipping if the surgeon was performing the cutting).  This ECD value will be 
ϱϱϯ
considered the baseline for these analyses.    Univariate models will be evaluated 
ϱϱϰ
first, and factors from those models with a p value <0.10 will then be evaluated in a 
ϱϱϱ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ
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multivariate model.  A final model will be constructed consisting of factors with a p 
ϱϱϲ
value <0.01 following a backwards selection process.      
ϱϱϳ
 
ϱϱϴ
Potential factors to evaluate include: 
ϱϱϵ
• Recipient factors 
ϱϲϬ
¾ 
preoperative diagnosis 
ϱϲϭ
¾ 
gender 
ϱϲϮ
¾ 
age 
ϱϲϯ
¾ 
race  
ϱϲϰ
¾ 
prior use of glaucoma medication 
ϱϲϱ
¾ 
prior glaucoma surgery (trabeculectomy, laser procedure) 
ϱϲϲ
¾ 
current smoker (at time of surgery) 
ϱϲϳ
¾ 
lens status (phakic, posterior chamber intraocular lens) 
ϱϲϴ
¾ 
Intraocular pressure (IOP)  
ϱϲϵ
 
ϱϳϬ
• Donor/graft factors 
ϱϳϭ
¾ 
eye bank determined screening ECD  
ϱϳϮ
¾ 
pre-operative CIARC determined ECD 
ϱϳϯ
¾ 
age 
ϱϳϰ
¾ 
gender 
ϱϳϱ
¾ 
race 
ϱϳϲ
¾ 
history of diabetes 
ϱϳϳ
¾ 
cause of death 
ϱϳϴ
¾ 
type of storage medium 
ϱϳϵ
¾ 
death to preservation time 
ϱϴϬ
¾ 
surgeon cut vs eye bank cut 
ϱϴϭ
¾ 
postcut thickness 
ϱϴϮ
¾ 
cut to surgery time 
ϱϴϯ
¾ 
observations during or after cutting 
ϱϴϰ
 
ϱϴϱ
• Surgical factors  
ϱϴϲ
¾ 
Insertion method 
ϱϴϳ
¾ 
Incision location 
ϱϴϴ
¾ 
Incision site 
ϱϴϵ
¾ 
Graft size 
ϱϵϬ
 
ϱϵϭ
• Postoperative factors  
ϱϵϮ
¾ 
Dislocation 
ϱϵϯ
¾ 
Rebubbling 
ϱϵϰ
¾ 
IOP  
ϱϵϱ
¾ 
Graft rejection 
ϱϵϲ
¾ 
Corneal thickness 
ϱϵϳ
 
ϱϵϴ
 
ϱϵϵ
 
ϲϬϬ
4.3 Effect of Preservation Time on Pre-Operative ECD – (Secondary Non-Protocol 
ϲϬϭ
Objective) 
ϲϬϮ
An ANCOVA model with CIARC graded pre-operative ECD as the dependent variable, 
ϲϬϯ
and adjusting for eye-bank-determined screening ECD, will be used to assess the effect 
ϲϬϰ

Wd^^ƚĂƚŝƐƚŝĐĂůŶĂůǇƐŝƐWůĂŶǀϰϲͲϮϭͲϭϲŶŽĂƉƉĞŶĚŝǆ
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of preservation time.  The time from preservation to surgery will be treated as a binary 
ϲϬϱ
variable.  
ϲϬϲ
 
ϲϬϳ

ϲϬϴ
5.0 Safety Analysis Plan 
ϲϬϵ
All reported adverse events will be tabulated by treatment group.  The main safety analysis will 
ϲϭϬ
involve tabulation of data by treatment group of events that could be considered possibly related to 
ϲϭϭ
the preservation time, including endophthalmitis, bacterial, fungal or parasitic corneal infection, or 
ϲϭϮ
any other events designated by the study group as possibly related to preservation time.   
ϲϭϯ
 
ϲϭϰ
Operative complications and procedures, post-operative complications and procedures (including 
ϲϭϱ
dislocation of donor, interface fluid, air injection), and abnormalities noted on ocular exam will also 
ϲϭϲ
be tabulated by treatment group to evaluate potential safety concerns.  
ϲϭϳ
 
ϲϭϴ
The efficacy analyses already outlined in this document, related to graft failure and ECDs, also 
ϲϭϵ
could be viewed as safety analyses.  Occurrences of following additional events during follow-up 
ϲϮϬ
will be tabulated by treatment group to assess potential safety concerns: 
ϲϮϭ
• IOP>25 mmHg (median and quartiles will also be presented) 
ϲϮϮ
• Corneal Thickness >750 microns (median and quartiles will also be presented) 
ϲϮϯ
• Definite signs of graft rejection 
ϲϮϰ
• Presence of stromal corneal vessels 
ϲϮϱ
• Presence of corneal scar or haze 
ϲϮϲ
• Epithelial defect >50% 
ϲϮϳ
• Donor stromal clarity = cloudy 
ϲϮϴ
• Recipient stromal clarity = cloudy 
ϲϮϵ
 
ϲϯϬ
6.0 Additional Tabulations and Analyses 
ϲϯϭ
The following will be tabulated according to treatment group: 
ϲϯϮ
• Baseline recipient demographic and clinical characteristics 
ϲϯϯ
• Donor characteristics 
ϲϯϰ
• Visit completion rate for each visit 
ϲϯϱ
• Additional post-operative study eye procedures(not part of the safety analysis), at each visit 
ϲϯϲ
• Baseline characteristics in cases with graft failure, cases with incomplete follow up without 
ϲϯϳ
graft failure, and cases with complete follow up without graft failure. 
ϲϯϴ
• Crosstabulation of immunizations or vaccinations versus signs of graft rejection, at each visit 
ϲϯϵ
• Protocol deviations 
ϲϰϬ
 
ϲϰϭ
7.0 DSMC Interim Analysis Plan  
ϲϰϮ
No formal interim analyses are planned towards demonstrating non-inferiority before the end of the 
ϲϰϯ
study since the recruitment period is planned to be short compared with the follow-up period and 
ϲϰϰ
since we believe it is imperative to have three years of follow up to assess non-inferiority.   
ϲϰϱ
 
ϲϰϲ
In addition to semi-annual review described in Section 5.2, the following plan for interim monitoring 
ϲϰϳ
for a potential recommendation of early stopping of enrollment has been established in conjunction 
ϲϰϴ
with the DSMC.  This plan is based on early donor failure rate and on the progress of recruitment.  
ϲϰϵ
• Rate of failure within the first 8 weeks:  Upon enrollment of the first 100 eyes, and then 
ϲϱϬ
quarterly thereafter (i.e., one review between each DSMC semi-annual meeting) the DMAC 
ϲϱϭ

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will evaluate the failure rate within the first 8 weeks (i.e. both the early failures and the 
ϲϱϮ
primary donor failures, as defined in Section 4.6) in each group and notify the DSMC who 
ϲϱϯ
will have the option of requesting additional information between the semi-annual reviews.  
ϲϱϰ
The DSMC may also request more frequent reviews at any time.  
ϲϱϱ
• Recruitment Progress:   Recruitment progress will be evaluated at the first two DSMC 
ϲϱϲ
meetings following initiation of recruitment.    If based on the current recruitment total and 
ϲϱϳ
recruitment trend over the previous 3 months, the projected timeline for the remaining 
ϲϱϴ
recruitment is more than 16 months at the 1st review or more than 12 months at the 2nd review, 
ϲϱϵ
the DSMC will discuss whether the study timeline can be met.  
ϲϲϬ
 
ϲϲϭ
8.0 Extension Analysis Plan  
ϲϲϮ
8.1 Background 
ϲϲϯ
CPTS Protocol Amendment V 4.0 7-1-15 extended follow up such that participants who are 
ϲϲϰ
willing to re-consent will continue to be monitored for annual protocol visits past the 3-year 
ϲϲϱ
timepoint, until at least 2017.  The same data will be captured from annual visits as well as 
ϲϲϲ
interim unscheduled visits as was done previously. The same study specific tests – pachymetry 
ϲϲϳ
and specular/confocal microscopy - will continue to be performed on the annual visits. All 
ϲϲϴ
participants will therefore have a last possible common endpoint rather than exiting individually 
ϲϲϵ
when they reach their 3 year post-operative visit; all visits will continue to be captured until 
ϲϳϬ
approximately the 2nd quarter of 2017.  
ϲϳϭ
Rationale: Continued follow-up of CPTS participants provides an excellent opportunity to gain 
ϲϳϮ
more information about many factors affecting DSAEK outcomes, including donor age and 
ϲϳϯ
preservation time. We estimate a maximum potential of 740 and 160 Year 4 and 5 visits, 
ϲϳϰ
respectively, if most participants re-consent. This additional longitudinal data will provide 
ϲϳϱ
important information on longer term DSAEK outcomes that are not necessarily impacted by 
ϲϳϲ
preservation time, although we will continue to assess that variable as well.   
ϲϳϳ
 
ϲϳϴ
Actual number 
of surgeries, 
spread into 
timing of when 
they occurred 
 
  
84% of enrolled 
are expected to 
reach 3 year 
90% of those 
reaching 3 year are 
expected to consent 
to CPTS extension  
  
  
Enrolled 
CPTS Grant 
  
Projected to 
reach 3 year 
visit 
Projected to consent 
to CPTS extension 
4 year visit 
5 year visit 
52 
Y4 
Q2 2015 
44 
39 
  
  
183 
Q3 2015 
154 
138 
  
  
197 
Y5 
Q4 2015 
165 
149 
  
  
187 
Q1 2016 
157 
141 
  
  
232 
Q2 2016 
195 
175 
37 
  
181 
Q3 2016 
152 
137 
131 
  
204 
Y6 
Q4 2016 
171 
154 
141 
  
94 
Q1 2017 
79 
71 
134 
  
 
Q2 2017 
  
  
167 
35 
 
Q3 2017 
  
  
130 
125 
 
ϲϳϵ
8.2 Objectives  
ϲϴϬ
The objective of the extension is to extend the 3 original study objectives to the 4 and 5 year 
ϲϴϭ
endpoints. 
ϲϴϮ

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8.3 Statistical Methods 
ϲϴϯ
The same statistical methods for all 3 study objectives as outlined above for analysis at 3 
ϲϴϰ
years will be extended to the 4 year and 5 year endpoints.  
ϲϴϱ
Baseline characteristics will be tabulated and compared between subjects who completed a 3 
ϲϴϲ
year visit and chose not to participate versus those who completed a 3 year visit and 
ϲϴϳ
consented to participate, stratified by preservation time group.  Subjects who dropped prior 
ϲϴϴ
to the 3 year visit for any reason (regraft, death, LTF, withdrew) will be excluded from this 
ϲϴϵ
comparison. 
ϲϵϬ
 
ϲϵϭ