Assessment of Theranostic Guided Riboflavin/UV-A Corneal Cross-linking for Treatment of Keratoconus
Phase: NA
Conditions: Keratoconus
Sponsor: Regensight

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STUDY PROTOCOL 
Assessment of theranostic guided riboflavin/UV-A corneal cross-linking  
for treatment of keratoconus 
 
 
 
ACRONYM: ARGO 
PROTOCOL NUMBER: RSKC001 
PROMOTER & SPONSOR: Regensight srl, Via Livenza 3, 00198 Rome (Italy) 
SPONSOR CONTACT: Dr. Marco Lombardo, C.E.O., mlombardo@regensight.com 
TRIAL TYPE: PRE-MARKET CLINICAL STUDY  
PROTOCOL VERSION: 1.0 - 28/06/2021 
REVISION HISTORY: 
1.0 
First emission 
 
SITES OF INVESTIGATION:  
• Azienda Ospedaliera Universitaria Careggi, Università di Firenze  
• Azienda Ospedaliera Universitaria Policlinico G. Martino, Università di Messina  
• Azienda Ospedaliera Universitaria Mater Domini – Università Magna Graecia di Catanzaro  
 
COORDINATOR SITE OF INVESTIGATION:  
• Azienda Ospedaliera Universitaria Mater Domini – Università Magna Graecia di Catanzaro  
 
INVESTIGATIONAL MEDICAL DEVICE: C4V CHROMO4VIS release sw 2.0.0 
MANUFACTURER: Regensight srl, Via Livenza n.3, 00198 Rome (Italy) 
 
 
 

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PROTOCOL APPROVAL and SIGNATURES 
Sponsor 
I declare that the protocol ARGO (Version 1.0 - 28/06/2021) contains all necessary information 
required for the conduct of the trial. 
Dr. Marco Lombardo – Chief Executive Officer (C.E.O.) 
       04/10/2021 
Signature 
 
 
xxxxxxxxxxxxxxxxxx 
Investigators’ Statement 
I have carefully read this protocol and agree to conduct the study in accordance with Good Clinical 
Practice and all applicable government regulations relevant to the use of new and approved 
therapeutic agents in human subjects.  
I agree that Regensight srl, its delegates and Regulatory Authorities have direct access to all study 
documentation.  
I agree to obtain the written Informed Consent from all participating subjects.  
I agree to maintain the confidentiality of all information received or developed in connection with this 
clinical trial. 
 
Prof. Rita Mencucci – Investigator 
____/_____/____ 
Signature 
 
 
 
 
Prof. Anna Maria Roszkowska – Investigator  
 
____/_____/____ 
Signature 
 
 
 
 
Prof. Vincenzo Scorcia  – Investigator  
 
____/_____/____ 
Signature 
 
 
 
 

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Declaration of the Sponsor on the sources and types of financial, material and other 
support  
The Sponsor loans, free-of-charge, the medical devices and the equipment required to perform 
treatment to the Investigators/Institutions for the entire duration of the trial. The free of charge supply 
of these materials does not influence the scientific, technical and procedural autonomy of the 
Investigators and Study staff.  
The medical devices used in this Study will be stored and handled according to the applicable Laws. 
The equipment will be handle with care and used exclusively in the clinical trial. 
The Sponsor will train the investigators on how to use these devices. The Sponsor undertakes the 
liability insurance required for each subject enrolled in the Study.  
 
 
 
 
 
 
 
 
 
 
 
Proprietary Notice: The concepts and information contained in this document or generated during 
the study are considered proprietary and may not be disclosed in whole or in part without the 
expressed, written consent of REGENSIGHT srl, except for eventual discussions with regulatory 
authorities, Ethical Committee or persons participating in the conduct of the study. 
Ethics Statement: The study is performed in compliance with good clinical practice (GCP), 
Declaration of Helsinki and regulations relevant to the use of new therapeutic agents in human 
subjects. 

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Annexes 
Annex 1 
Case Report Form (CRF) 
Annex 2 
General instructions for compiling CRF 
Annex 3 
Research Subject Information and Consent Form (in italiano) 
Annex 3 bis 
Personal data treatment (in italiano) 
Annex 3 ter 
Letter to general medical practitioner & patient advice (in italiano) 
Annex 4 
List of drop out participant 
Annex 5 
3-months report 
Annex 6 
Interim report (6-months) 
Annex 7 
Final report 
Annex 8 
Investigator’s Brochure 
Annex 9 
User Manual – For Research Use Only (in italiano) 
Annex 10 
Accordo di Studio Clinico 
Annex 11 
Assicurazione Sanitaria 
Annex 12 
Lettere divulgazione studio clinico  
 
 

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Rationale of the Study and Risk/Benefit analysis 
Keratoconus is a naturally-occurring ocular condition characterized by progressive thinning and 
steepening of the central cornea, resulting in corneal optical irregularities with increasing myopia, 
irregular astigmatism, corneal opacity and consequential loss of visual acuity. Clinical onset of the 
disease begins in the second decade of life and has significant social impact due to its progression 
towards vision loss; indeed, keratoconus still represents the primary cause of corneal transplantation 
in young adults globally. 
Riboflavin/UV-A corneal cross-linking is a procedure used to biomechanically stabilize the weak 
cornea in keratoconus and to slow down or halt the clinical progression of this disease. The 
procedure has been performed in Europe since 2003, thereafter the procedure has been adopted in 
eastern countries and United States. The standard corneal cross-linking protocol, also known as 
Dresden protocol, consists in removing the epithelium and administering a riboflavin ophthalmic 
solution onto the corneal stroma for 30 minutes; afterwards the cornea is irradiated by a UV-A light 
device using 3mW/cm2 power density for 30 minutes, with a total delivered energy of 5.4 J/cm2. 
According to the systematic review and meta-analysis studies, the standard cross-linking protocol is 
effective (where efficacy is indicated as stabilization or flattening of the maximum simulated 
keratometry value, Kmax) in almost 70% of treated cases, with a failure rate ranging from 8% to 33% 
one year after surgery. However, epithelial removal is the predisposing factor to the most frequent 
and major complications of the standard protocol, which include ocular pain, corneal haze, transient 
corneal edema in virtually all cases, and some severe adverse events, such as corneal infections, 
corneal melting, and corneal scarring, which may cause vision loss. For this reason, any 
improvement, which can increase the efficacy and minimize the risks of the gold standard surgical 
technique is highly desirable. Several transepithelial treatment protocols have been developed in 
order to minimize risks, nevertheless their clinical efficacy in comparison with the standard protocol 
still remains object of debate.  
Theranostics is an emerging therapeutic paradigm that enables monitoring of image-guided  
therapy in clinic through the use of a theranostic module that makes use of real-time non-invasive 
molecular analysis of the tissue being treated to achieve optimal treatment outcomes in the 
management of disease. 
This is a clinical study consisting of a study arm to assess the performance of the theranostic 
software module (Research Use Only) of a CE marked (CE1936) UV-A medical device, C4V 
CHROMO4VIS™, in order to validate its use for image-guided corneal cross-linking treatment of 
keratoconus and corneal ectasia. The study hypothesis is that theranostic-guided riboflavin/UV-A 
corneal cross-linking with the C4V CHROMO4VIS™ system is safe and can estimate treatment 
efficacy during operation, regardless of treatment protocol, i.e., either with or without epithelial 

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removal. The software module is able to measure the concentration of riboflavin into the cornea 
during treatment and to provide the surgeon with an objective assessment of treatment efficacy. The 
scope of this study is to validate the theranostic imaging biomarker score by assessing the change 
of corneal topography Kmax value at 1-year postoperatively. The 1-year follow-up is long enough to 
provide scientific evidence of the safety and efficacy of the theranostic UV-A medical device in 
question. A pre-operative examination will ensure that every interested and willing participant fulfils 
the inclusion criteria of this study. Post-operative examinations will be carried out after 1 week, 1 
month, 3 months, 6 months and 12 months.  
The present study can start treatment of subjects only after an Independent Ethical Committee 
written approval; it is governed by three guiding principles: safety, ethical conduct, and efficiency. 
Subject safety guides study design by minimizing the number of participants exposed to serious 
adverse events.  
 
 
 
 

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Abbreviations 
AE 
Adverse Event 
CDVA 
Corrected Distance Visual Acuity 
C4V CHROMO4VIS™ 
Theranostic UV-A medical device  
C4V  
Theranostic UV-A medical device acronym 
CFR 
Code of Federal Regulation  
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CTA 
Clinical Trial Authorization 
CV 
Curriculum Vitae 
CXL 
Corneal cross-linking 
D 
Diopter 
EpiOFF 
Epithelium off 
EpiON 
Epithelium on or transepithelial 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation 
GMP 
Good Manufacturing Practice 
ICH 
International Council for Harmonization 
IEC 
Independent Ethical Committee 
Kmax 
Maximum Simulated Keratometry 
LED 
Light Emitting Diode 
LogMAR 
Logarithmic Minimum Angle of Resolution 
RitSight™ 
0.22% riboflavin eye drop 
SAE 
Serious Adverse Event 
UCVA 
Uncorrected Distance Visual Acuity 
UV-A 
Ultraviolet light A 
 
 
 

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Table of Contents 
Synopsis ........................................................................................................................................................... 11 
Background and State-of-the-Art .................................................................................................................... 20 
Riboflavin/UV-A corneal cross-linking for the treatment of keratoconus .................................................. 21 
Investigational medical device ........................................................................................................................ 24 
System Description ...................................................................................................................................... 24 
Major Components ...................................................................................................................................... 24 
Technical Features ....................................................................................................................................... 26 
Optical Radiations ........................................................................................................................................ 26 
Environmental Conditions ........................................................................................................................... 26 
Electrical Data .............................................................................................................................................. 27 
On-Board PC Specifications ......................................................................................................................... 27 
Touch Screen ............................................................................................................................................... 27 
System Mechanical Features ....................................................................................................................... 28 
Main UV-A medical device operation .......................................................................................................... 28 
UV-A irradiation plans ................................................................................................................................. 29 
UV-A irradiation types ................................................................................................................................. 29 
Contraindications ........................................................................................................................................ 30 
Adverse Events ............................................................................................................................................ 31 
Precautions .................................................................................................................................................. 31 
List of International Standards .................................................................................................................... 32 
Assessing the theranostic software module 2.0.0 ...................................................................................... 34 
Theranostic-guided UV-A irradiation plans ................................................................................................. 34 
Theranostic-guided CXL operation sequence .............................................................................................. 34 
Pre-clinical studies on safety of the UV-A medical device .......................................................................... 36 
Pre-clinical studies on efficacy of the theranostic UV-A medical device .................................................... 39 
Sterile ophthalmic riboflavin solution ............................................................................................................. 41 
Product Description ..................................................................................................................................... 41 
Pre-clinical studies on safety of the 0.22% riboflavin ophthalmic solution ................................................ 43 
Study Plan ........................................................................................................................................................ 44 
Aim ............................................................................................................................................................... 44 
Type of study and duration ......................................................................................................................... 44 
Study Participants ........................................................................................................................................ 44 
Study Arm .................................................................................................................................................... 45 
Discontinuation of contact lens wear .......................................................................................................... 45 

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Outcome Measures and Assessments ......................................................................................................... 45 
Study Visits .................................................................................................................................................. 46 
Visit 1 – Baseline .......................................................................................................................................... 46 
Treatment (day 0) ........................................................................................................................................ 47 
Visit 2, 3, 4 and 5 – Follow up (day 7, day 30, day 90 and day 180 respectively) ....................................... 47 
Visit 6 – Follow up (day 360) ....................................................................................................................... 48 
Dropouts ...................................................................................................................................................... 48 
Treatment ........................................................................................................................................................ 49 
Patient Counseling Information .................................................................................................................. 50 
Study Product Packaging and Storage ......................................................................................................... 51 
Test Article Accountability ........................................................................................................................... 51 
Study assessments ........................................................................................................................................... 52 
Demographic Data/Medical History ............................................................................................................ 52 
Efficacy Assessments ................................................................................................................................... 52 
Safety Assessments ..................................................................................................................................... 52 
Primary outcome measure .......................................................................................................................... 52 
Secondary outcome measures .................................................................................................................... 53 
Other outcome measures ............................................................................................................................ 53 
Adverse Events ............................................................................................................................................ 54 
Reporting adverse events ........................................................................................................................ 54 
Eliciting Adverse Event Information ........................................................................................................ 55 
Adverse Event Reporting ......................................................................................................................... 55 
Assessment of Causality .......................................................................................................................... 56 
Assessment of Severity ............................................................................................................................ 56 
Action taken to Adverse Event ................................................................................................................ 57 
Adverse Event treatment required .......................................................................................................... 57 
Outcome of Adverse Event ...................................................................................................................... 57 
Serious Adverse Event Reporting ............................................................................................................ 57 
Adverse Event Monitoring ....................................................................................................................... 58 
List of adverse events reported in the specialized literature after CXL treatment ................................. 58 
Summary of adverse events on total 9607 cases treated by CXL procedures (ref. bibliography) .............. 58 
Treatment strategy for adverse events ................................................................................................... 59 
Statistical Considerations and Data Analysis ................................................................................................... 60 
Stratification, Allocation and Enrolment ..................................................................................................... 60 
Synopsis on disposition of participants ....................................................................................................... 60 

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Sample size .................................................................................................................................................. 60 
Statistical methodology ............................................................................................................................... 61 
Population to be analysed ........................................................................................................................... 61 
Ethical and Regulatory Issues .......................................................................................................................... 62 
Good Clinical Practice (GCP) for Clinical trials ............................................................................................. 62 
Independent Ethics Committee approval .................................................................................................... 63 
Informed consent ........................................................................................................................................ 63 
Risk Assessment and Management ............................................................................................................. 64 
Sites of Investigation ....................................................................................................................................... 66 
Investigator qualifications ........................................................................................................................... 67 
Sponsor ............................................................................................................................................................ 68 
Administrative Rules ........................................................................................................................................ 69 
Clinical data handling .................................................................................................................................. 69 
Case Report Forms ...................................................................................................................................... 69 
Reports ........................................................................................................................................................ 70 
Study Reporting Requirements ................................................................................................................... 71 
Monitoring of the Study .............................................................................................................................. 71 
Monitoring Plan ........................................................................................................................................... 72 
Inspection of Records .................................................................................................................................. 73 
Study Record Retention ............................................................................................................................... 73 
Quality Assurance ........................................................................................................................................ 73 
Confidentiality ............................................................................................................................................. 74 
Personal data protection ............................................................................................................................. 74 
Modification of the Protocol ....................................................................................................................... 74 
Protocol Violations and Deviations ............................................................................................................. 75 
List of major deviations/violation ................................................................................................................ 75 
Financial Disclosure and Obligations ........................................................................................................... 76 
Study Conduct ............................................................................................................................................. 76 
Recruitment strategies ................................................................................................................................ 76 
Publications ................................................................................................................................................. 77 
Study Termination ....................................................................................................................................... 77 
References ....................................................................................................................................................... 78 
Appendix 1 - Methodology for sample size calculation .................................................................................. 82 
 

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Synopsis 
Study Population 
Ages eligible for study: from 18 years old to 40 years old. 
Genders eligible for study: all genders. 
Accepts Healthy Volunteers: No. 
Inclusion Criteria 
Diagnosis of progressive keratoconus.  
The criterion to determine progression of keratoconus is based on providing at least one of the 
following types of evidence:  
• 
at least two Placido disk corneal topography measurements showing at least +1.00 D 
steepening of the Kmax value in the last year or longer interval period. 
• 
at least two manifest refraction measurements showing at least -0.50 D change in spherical 
equivalent refraction in the last year or longer interval period. 
• 
at least two central corneal thickness (CCT) measurements showing at least -10 µm change in 
in the last year or longer interval period. 
Exclusion Criteria 
- 
Anterior corneal curvature steeper than 63 D; 
- 
Corneal thickness thinner than 400 µm; 
- 
Corneal scarring; 
- 
Descemetocele;  
- 
History of herpetic keratitis; 
- 
Concomitant eye diseases; 
- 
Inflammatory eye diseases; 
- 
Glaucoma; 
- 
Cataract; 
- 
Nistagmus; 

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- 
Pregnancy; 
- 
Breast feeding. 
Ocular history 
From each study participant a complete medical and ocular history shall be obtained. 
• 
Family history of keratoconus. 
• 
History of dermatologic pathology. 
• 
History of atopy or allergy. 
• 
History of contact lens wear. 
• 
History of eye rubbing. 
• 
History of medication. 
Study objective 
The scope of the study is to evaluate a novel modality of riboflavin/UV-A corneal cross-linking based 
on theranostics aiming at improving predictability of treatment efficacy for better patient care. The 
objective of the study is to validate the theranostic imaging biomarkers by assessing the mean 
change of Kmax value at 12 months after riboflavin/UV-A corneal cross-linking for the treatment of 
keratoconus.  
Study design 
Subjects with a diagnosis of progressive keratoconus will be evaluated for suitability as a candidate 
for riboflavin/UV-A corneal cross-linking. Participants will be evaluated at baseline, day 0 (treatment), 
day 7, day 30, day 90, day 180 and day 360 after treatment. Informed consent will be obtained from 
each subject before performance of any required procedures.  
Eye examination 
Each study participant will undergo at baseline and at each follow-up visit: 
• 
Uncorrected Distance Visual Acuity (UDVA) with ETDRS chart. 
• 
Corrected Distance Visual Acuity (CDVA) with ETDRS chart. 
• 
Manifest Spherical Equivalent Refraction (MSER) with ETDRS chart. 

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• 
Maximum Simulated Keratometry (Kmax) and Central Corneal Thickness (CCT) with combined 
corneal topography and pachymetry. 
• 
Endothelial Cell Density (ECD) with corneal specular microscopy. 
• 
Slit-lamp examination of the cornea, anterior chamber and lens. 
Study arm 
One study arm receiving riboflavin/UV-A corneal cross-linking with either standard, Epi-OFF, or 
transepithelial, Epi-ON, treatment protocol. Only one eye of each participant is designated as the 
study eye. 
Stratification, Allocation and Enrolment 
Eligible participants will be stratified with allocation ratio 1:1 into either treatment protocol using a 
computer-generated stratification plan with blocks. Two different blocks are created, which include 
eyes with Kmax steeper or flatter than 54.0 D to allocate patients with comparable baseline Kmax values 
in either treatment protocol.  
This is a multi-center clinical trial. The stratification code is given to each local site of investigation 
by the central monitoring site of the Sponsor after the participant has been considered eligible to the 
study and has signed the informed consent. The enrolment is competitive. The period for enrollment 
includes the four months after study protocol approval by the IEC of the local site of investigation.  
Sample size 
This study aims to validate the theranostic imaging biomarkers by assessing the change of maximum 
keratometry (Kmax) after corneal cross-linking treatment (CXL). Based upon a systematic literature 
review, the study hypothesis is that CXL treatment can averagely flattening the Kmax value by -1.05 
D (SD: ±0.80 D) at 1-year after CXL. To this scope, expecting a response rate of 50% of cases that 
will reach the threshold of -1.05 D of the Kmax value, a sample size of 42 patients achieves 91% 
power to detect a difference of 0,2500 between the area under the ROC curve (AUC) under the null 
hypothesis of 0,6000 and an AUC under the alternative hypothesis of 0,8500 using a two-sided z-
test at a significance level of 0,05000. Considering a ≤20% drop-out rate (determined from 

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systematic literature review on controlled and open clinical studies on standard and epithelium-on 
CXL procedures), a number of 50 participants (25 per treatment protocol) is allowed to be enrolled 
in the study. 
Products’ description 
The test articles to use in this study include two CE marked medical devices, such as the 
investigational one, herein the C4V CHROMO4VIS™ (CE1936) and the 0.22% riboflavin ophthalmic 
solution, herein the RitSight™ eye drop (CE0477), which must be applied onto the cornea before 
UV-A irradiation to protect the internal ocular structures from excessive UV-A light radiation. 
The C4V CHROMO4VIS™ is a CE marked class IIb electronic UV-A medical device, which delivers 
ultraviolet light (365 nm wavelength) in a circular area onto the cornea. It is a portable system with 
articulating arm to allow movement of the system for alignment of the UV-A beam onto the patient’s 
cornea. UV irradiance and irradiation time at the cornea are controlled by an onboard computer 
system. The medical device is equipped with the theranostic software (for research purpose only) 
that is the object of validation in this study. The RitSight™ is a CE marked sterile class IIa medical 
device consisting of a 0.22% riboflavin enriched solution for effective penetration of riboflavin into 
the corneal tissue to protect the inner ocular structures during UV-A irradiation. 
Treatment 
Corneal cross-linking is performed with either the corneal epithelium intact or de-epithelialized in 
patients with progressive keratoconus. The ophthalmic solution RitSight™ is applied onto the cornea 
and its concentration into the cornea monitored by C4V CHROMO4VIS™ system with theranostic 
software module (v. 2.0). After the pre-set time for application of riboflavin onto the cornea is ended 
(i.e., 15 min. for epi-off CXL and 20 min. for epi-on CXL protocol), the cornea is illuminated by the 
C4V CHROMO4VIS™ system using 5.4 J/cm2 UV-A energy dose (10 mW/cm2 for 9 minutes).  
 
 
 

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Primary End Point and Outcome Measure 
Time point 12 months: validation of the theranostic imaging biomarkers. 
Validation of the combined use of the theranostic imaging biomarkers. True positives, false positives, 
true negatives and false negatives were evaluated in order to determine the ability of the theranostic 
imaging biomarkers, including both the riboflavin score and theranostic score, to predict the 
propensity of CXL treatment in flattening the Kmax value (D) by more than 0.1 D at 12 months 
postoperatively. True positives, false positives, true negatives and false negatives will be evaluated. 
Secondary Outcome Measures 
• Efficacy: Change of Maximum Keratometry (Kmax) assessed by corneal topography at 12-months. 
• Safety: change of Endothelial Cell Density (ECD) assessed by specular microscopy at 12-months. 
Exploratory Outcome Measures 
• Change of Uncorrected Distance Visual Acuity (UDVA) assessed by ETDRS at 12-months. 
• Change of Corrected Distance Visual Acuity (CDVA) assessed by ETDRS at 12-months. 
• Change of Manifest Refraction assessed by ETDRS at 12-months. 
• Change of primary and secondary outcome measures in either stratification group at 12-months. 
Adverse events 
Any adverse event (AE) occurring in a participant enrolled into the clinical trial shall be reported in 
detail on the CRF according to the ICH Guidelines and the MDCG 2020-10-2 Guidance safety report 
form. The Medical Dictionary for Regulatory Activities (MedDRA) is used to code all AEs. 
Study Records/Source Documents/Personal Data protection 
Adequate records (Case Report Forms) will be maintained for the study including subject medical 
and surgical records, test reports, signed informed forms, and information about subject 
discontinuation. All original documentation will remain at the investigative sites. Study data that are 
generated electronically (e.g. corneal topography etc.) will be printed and retained in the study files. 
A unique identifier will be assigned by the Investigators to each trial subject to protect the subject's 
identity.  

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Dropouts/Lost-to-follow-up/withdrawal 
Participants may drop out at any time during the study. Based on the scientific literature and 
considered the 1-year follow-up, a ≤20% dropout may be expected in the study. A total number of 
50 participants is therefore allowed to be enrolled in the study. 
Statistical Methods 
Descriptive statistics will be used to summarize the data: numerical variables will be summarized as 
mean and standard deviation or median and interquartile range, respectively, according to the 
distribution of the data; categorical variables will be represented as frequencies and proportions. 
Student’s t-test or Wilcoxon test will be applied to compare continuous variables, while Fisher's exact 
test or Chi-Squared test will be used to analyze categorical variables. ROC curve analysis will be 
used to evaluate cut-off values for the theranostic imaging biomarker scores in predicting the 
propensity of CXL treatment to flatten the Kmax value at 12-months. The cut-off value for the 
biomarker scores will be determined by optimizing the Youden index. Moreover, sensitivity, 
specificity, proportion of correctly classified eyes and positive predictive value (PPV) positive 
predictive values (PPV) and false negative rate (FNR) and confidence intervals will be calculated.  
The study set a minimum threshold of 85% for the theranostic software module’s accuracy and 
precision in predicting the propensity of CXL to halt disease progression (intended as more than 0.1 
D Kmax flattening) at 1 year in the study population (per protocol population). 
Statistical significance will be set at 0.05. All the analysis will be performed using the statistical 
software R (latest version available). 
Data Analysis 
Data will be summarized using descriptive statistics and graphical presentations.  
Numerical variables will be summarized as mean and standard deviation (SD) and as median and 
interquartile range (IQR). Categorical variables will be represented as frequencies and proportions. 
For numerical variables, paired Student t-test or Wilcoxon signed-rank test will be used to compare 
the distributions preoperatively and 12-months postoperatively. Bonferroni correction will be 

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applied to analysis of exploratory outcome measures of stratification groups. For all reported AEs, 
the data shall be presented with events listed in order of decreasing frequency. 
Sites of Investigation 
• Azienda Ospedaliera Universitaria Careggi, Università di Firenze  
• Azienda Ospedaliera Universitaria Policlinico G. Martino, Università di Messina  
• Azienda Ospedaliera Universitaria Mater Domini, Università Magna Graecia di Catanzaro  
Coordinator Site of Investigation 
• Azienda Ospedaliera Universitaria Mater Domini, Università Magna Graecia di Catanzaro  
List of principal investigators 
Prof.ssa Rita Mencucci, ophthalmologist (UniFI) 
Prof.ssa Anna Maria Roszkowska, ophthalmologist (UniME) 
Prof. Vincenzo Scorcia, ophthalmologist (UniCZ) 
Declaration of the Sponsor on the sources and types of financial, material and other support  
The Sponsor has provided the clinical investigators and Site of Investigations with the Protocol 
(including Case Report Forms), the investigator’s brochure, confidentiality, personal data safety and 
sponsor agreements, and any other documentation, which is necessary to complete the Study.  
The Sponsor has verified in local audit that each clinical center has a designated clinical area and 
equipment as well as that the investigators are qualified and experienced to perform the present 
Study.  
The Sponsor loans, free-of-charge, the medical devices and the equipment required to perform 
treatment to the Investigators/Institutions for the entire duration of the trial. The free of charge supply 
of these materials does not influence the scientific, technical and procedural autonomy of the 
Investigators and Study staff.  The medical devices used in this Study will be stored and handled 
according to the applicable Laws. The equipment will be handled with care and used exclusively in 
the clinical trial. The Sponsor will train the investigators on how to use these devices. The Sponsor 
undertakes the liability insurance required for each subject enrolled in the Study.  

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Regulatory aspects and Funding Sources 
This clinical investigation is conducted in accordance with the provisions of articles 62 to 80 and 
Annex XV of the Regulation (EU) 2017/745 as part of the clinical evaluation for conformity 
assessment purpose of the UV-A theranostic medical device C4V CHROMO4VIS®. The object of 
the clinical investigation is the validation of the theranostic software module of a CE certified UV-A 
medical device for the treatment of pathological conditions of the cornea. The device in question 
conforms to the general safety and performance requirements apart from the aspects covered by 
the clinical investigation; with regard to those aspects, every precaution has been taken to protect 
the health and safety of the study participants.  
 
 
 
 

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Study protocol overview 
 
Baseline Visit 1
Patient not eligible
Visit 2 (day 7)
Visit 3 (day 30)
Visit 4 (day 90)
Stratification.
Treatment
(day 0)
Visit 5 (day 180)
Visit 6 (day 360).
End of study

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Background and State-of-the-Art 
Keratoconus is a degenerative corneal disease characterized by tissue weakening and thinning, 
which lead to corneal bulging (corneal ectasia). The incidence of keratoconus varies between 1:300 
and 1:2000 depending upon geographical location (it is more frequent in the south regions of the 
world). Prevalence of keratoconus is estimated between 0.5% and 3% depending upon geographical 
location. The etiopathogenesis of keratoconus is multifactorial; familiarity, which is found in 15% - 
18% of patients highlights the importance of genetics in the pathological pathway of keratoconus; 
beyond the genetic heterogeneity of keratoconus, the importance of various environmental factors 
(UV, atopy), which are able to trigger or accelerate the progression of corneal degeneration, is 
emphasized in the pathogenesis of the disease.1,2 The anatomical alterations of the corneal stroma 
in keratoconus have been correlated to a biomechanical tissue weakening. A number of 
experimental and clinical studies has shown decreased stiffness and altered viscoelasticity in 
keratoconus; the main theory is that tissue bulging is caused by inability of the keratoconic cornea 
to resist the physiologic intraocular pressure levels. 
Clinical onset of keratoconus begins in the second or third decade of life and involves a progressive 
vision loss caused by irregular astigmatism, induced myopia, and in more advanced cases, corneal 
scarring. Due to the young age of onset, keratoconus may have a significant negative impact on the 
quality of life of patients. Although contact lenses can provide the majority of patients with adequate 
visual quality, they are not able to slow down disease progression, which results in progressive vision 
loss. 
Treatment of keratoconus includes the use of eye glasses or contact lenses to improve visual acuity. 
The surgical treatment of keratoconus includes corneal cross-linking, which aims at stabilizing the 
structure and tissue biomechanics and halting disease progression. In more advanced cases, 
corneal transplantation is required to replace the degenerated corneal tissue for restoring visual 
function. However, corneal transplantation is not a permanent solution to visual disturbance 
occurring in keratoconus, due to the risk of postoperative complications, such as infections, tissue 
rejection, elevated and irregular astigmatism, etc. Corneal transplantation at a young age has a 9-
16% risk to be replaced in a lifetime, and the risk of rejection is persistent throughout life. For these 
reasons, a safe and effective intervention, such as corneal cross-linking with riboflavin and UV-A, 
which could stop disease progression would benefit at large the quality of life of millions of people in 
the world. 
 
1 Gordon-Shaag A, Millodot M, Shneor E, Liu Y.  The Genetic and Environmental Factors for Keratoconus. Biomed Res Int 2015; 2015: 795738. 
2 Cheung I, McGhee C, Sherwin T. A new perspective on the pathobiology of keratoconus: interplay of stromal wound healing and reactive species-
associated processes. Clin Exp Optom 2013; 96: 188-196. 

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Riboflavin/UV-A corneal cross-linking for the treatment of keratoconus  
Riboflavin/UV-A corneal cross-linking (CXL) is a treatment procedure aiming at slowing down or 
halting disease progression. The treatment consists in the application of riboflavin (vitamin B2) onto 
the cornea and subsequently in the illumination of the corneal tissue by ultraviolet light (UV-A).3 The 
riboflavin has the function to protect the corneal endothelium and inner ocular structures from 
excessive UV-A light exposure. Authors4 have demonstrated that CXL induces both a stiffening of 
the corneal stroma (measured as an increase in the elastic modulus of Young) and a significant 
decrease of viscoelasticity (measured as reduction of hysteresis). By translating the experimental 
results to the clinic, the increased corneal stiffness contributes to the stabilization effect on disease 
progression thanks to a greater tissue resistance to the physiologic intraocular pressure. There are 
likely other biological mechanisms that may contribute to improving treatment efficacy in the long 
term, such as the production of collagen proteins and stromal matrix components by the stromal 
keratocytes, which are regenerated from resident cell population of the posterior stroma after 
treatment.   
The treatment is performed on an outpatient basis under topical anesthesia; after surgery, the patient 
is prescribed topical medical therapy with antibiotics or antiseptics, anti-inflammatory and lubricant 
eye drops for a few weeks or months. In some cases, a therapeutic contact lens, which is removed 
within a few days after surgery, may be applied onto the operated eye. 
Riboflavin/UV-A corneal cross-linking has been performed in Europe since 2003, thereafter the 
procedure has been adopted in eastern countries and United States. Since the first clinical report on 
CXL published in 2003, over 800 scientific articles have been published discussing the clinical 
outcomes of the procedure for the management of keratoconus and secondary corneal ectasia. 
The standard corneal cross-linking protocol, also known as Dresden protocol, consists in removing 
the epithelium and administering a riboflavin solution onto the corneal stroma for 30 minutes; 
afterwards the cornea is irradiated by an UV-A light with 3mW/cm2 power density for 30 minutes, 
with a total delivered energy of 5.4 J/cm2. According to the current literature evidences, the standard 
cross-linking protocol is effective (where efficacy is indicated as stabilization or flattening of the 
maximum simulated keratometry value, Kmax) on almost 70% of treated cases, with a failure rate 
ranging from 8% to 33% one year after surgery. In general, the postoperative clinical course of the 
Dresden protocol shows a reduction in the corrected visual acuity during the first three months after 
treatment, a worsening of the keratometric topographic indices and a reduction in the central corneal 
thickness up to six months after treatment. The improvement of all clinical and instrumental 
 
3 Lombardo M et al. Interaction of ultraviolet light with the cornea: clinical implications for corneal crosslinking. J Cataract Refract Surg 2015; 
41(2):446-459. 
4 Beshtawi IM et al. Biomechanical properties of corneal tissue after ultraviolet-A-riboflavin crosslinking. J Cataract Refract Surg 2013; 39(3): 451-462.  

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biomarkers, in case of therapeutic success, becomes evident by the sixth postoperative month 
onwards.  
The simulated keratometry of the cone apex (Kmax or maximum keratometry index) is the most used 
index to demonstrate the progression of keratoconus and the efficacy of the corneal-cross linking. 
From a thorough literature review assessing clinical data on 6.995 cases treated by corneal cross-
linking with a UV-A medical device for the indication of use, the average change of Kmax has been -
1.05±0.80 D after 1 year follow-up, showing that the treatment is effective for stabilizing the cornea, 
which has been weakened by disease or laser surgery. These clinical data can be considered 
clinically significant when compared with the natural history of the disease, which induces an 
average Kmax steepening of +0.9 D per year. The bibliographic references revised to determine 
significance of statistical data in this clinical study can be found in Appendix 1 - Methodology of 
sample size calculation. 
By revising the clinical data on the efficacy of the gold standard protocol, i.e., the Dresden protocol, 
the Kmax decreases by -1.0 D and -1.7 D in almost 50% of cases at 1-year postoperatively; the 
corrected visual acuity (CDVA) on average stabilizes (mean change of -0.05 LogMAR in 50% of 
cases) and the manifest refraction on average changes by +0.50 D (in general, the reduction of the 
myopic sphere equivalent is between +0.2 D and +0.7 D) in the same period. The central corneal 
thickness on average remains almost unchanged (+4 μm) compared to the preoperative period at 1 
year after treatment. However, epithelial removal is the predisposing factor to the most frequent and 
major complications of the standard protocol, such as infectious keratitis, sterile corneal infiltrates, 
corneal scarring, which may cause vision loss.5  An additional risk factor in the standard protocol is 
the treatment of corneas with a central corneal thickness lower than 390 μm, due to the risk of 
inducing phototoxic damage to endothelial structures. 
In the last decade, an increasing number of treatment protocols have been developed in order to 
avoid epithelial removal and to reduce treatment time. Currently, CXL can be performed according 
to various treatment protocols, which are classified into two main groups, i.e., the protocols that 
require corneal de-epithelialization (also called EpiOFF) and those that do not require epithelial 
removal (also called EpiON or transepithelial protocols). Each type of surgical protocol, both EpiOFF 
and EpiON, in turn may vary according to the method and time of riboflavin application and the 
method and time of UV-A irradiation of the cornea. 
Currently, CXL is well established to treat progressive keratoconus and tens of thousands of 
treatments are performed yearly worldwide. Half of patients claim to have their quality of life much 
more improved after corneal cross-linking treatment and the procedure has proven to be a valid 
 
5 Alio JL et al. Corneal cross-linking and infectious keratitis: a systematic review with a meta-analysis of reported cases. J Ophthalmic Inflamm Infect 
2013;3(1):47. 

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therapy in halting keratoconus progression and drastically reducing the need of keratoplasty for 
thousands patients worldwide.  
Although CXL has been established as primary surgical option to treat progressive keratoconus, 
there is no current option to provide predictable outcomes and to minimize risk and improve efficacy 
for either the standard or the transepithelial protocol. The scope of the present clinical study is to 
assess the performance of a novel modality for riboflavin/UV-A corneal cross-linking using a 
theranostic UV-A medical device. Theranostics is going to introduce a novel methodology for 
targeting the precise therapeutical dose of riboflavin and UV-A into the corneal tissue in order to 
improve predictability of corneal cross-linking for halting disease progression. 
 

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Investigational medical device 
C4V CHROMO4VIS™ (software version 2.0) 
 
System Description 
The C4V CHROMO4VIS™ is an electronic medical device, which delivers ultraviolet light (365 nm 
wavelength) in a circular spot onto the area of the cornea to treat after a riboflavin ophthalmic solution 
has been properly applied. Emitted UV irradiance and energy dose are controlled by an on-board 
computer system.  
 
Major Components 
The major components of the C4V CHROMO4VIS system are:  
1. Optics head, including the UV-A, blue, yellow-green and orange LED sources, the iris 
aperture, the camera, Placido disk and an electronic board. 
2. Touch screen display with user interface.   
3. Stand, articulating arm and serving tray (accessory). 
4. Wheeled cart, including the AC/DC power supply and electronic boards. 
5. Foot switch. 
The major components of the system are described in detail as follows: 
1. The optics head houses an electronic board and the UV-A, blue, yellow-green and orange 
light sources, the iris aperture, the Placido disk and the camera: 
• 
UV-A light radiation centered at 365 nm wavelength with power density in the range 
between 3 mW/cm2 and 40 mW/cm2; 
• 
blue light radiation at a wavelength of 465 nm and power density ≤160 µW/cm2;  
• 
yellow-green light radiation at a wavelength of 572 nm and power density ≤50 µW/cm2; 
• 
orange light radiation at a wavelength of 605 nm; 
• 
a variable iris aperture is mounted in the UV-A irradiation beam path to provide a uniform 
circular area of irradiation at the treatment plane. The aperture is electronically controlled 
and can vary the UV-A beam diameter from 5 mm to 9 mm; 
• 
Placido disk to focus the blue light, and accordingly, the UV-A light beam onto the cornea 
to be treated.  
2. The treatment parameters (riboflavin dosing, iris aperture and UV-A irradiation) are selected 
and confirmed through the user interface touch screen display. 

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3. The C4V CHROMO4VIS system is a portable system with an articulating arm to allow 
movement of the system in medical areas for treatment of a patient. A serving tray 
(accessory) may be added to support medical and surgical supplies items.  
4. The power supply of C4V CHROMO4VIS system is managed by an electronic board, which 
provides power through an AC/DC switch power supply (100 Watt/12 Volt output) compliant 
with IEC60601-1.  
5. The treatment parameters can be confirmed by pressing the foot switch. 
 
 
Figure - Overview Illustration of the C4V CHROMO4VIS system. 
 
 
 

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Technical Features 
FEATURE 
TREATMENT 
LIGHT SOURCE 
UV irradiance 
UV irradiance 
From 3 mW/cm2 to 40 mW/cm2. 
UV-A 
UV exposure time 
From 2 minutes to 30 minutes. 
UV emission 
Continuous and intermittent (pulsed 
mode with on time and off time of 1 
second). 
Measured points 
Over 6.000 points. 
Corneal coverage 
From 5 mm up to 9 mm (step 1 mm); 
circular shape produced by a 12 blades 
iris. 
Focusing system 
Guided focus through Placido disk 
projecting rings for confirmation in x, y 
and z direction. 
Blue LED 
Power 
monitoring 
Double check system 
Continuous, 
on-board 
monitoring 
current and voltage across the UV-A 
LED light as well as using one 
independent dedicated UV-A sensor. 
N/A 
 
Optical Radiations 
FEATURE 
LIGHT SOURCE 
WAVELENGHT 
POWER DENSITY 
ON EYE 
Central fixation LED 
Yellow-green LED 
572 nm 
≤50 µW/cm2 
Illumination of Placido disk 
for alignment and focusing 
Blue LED 
470 nm 
≤160 µW/cm2 
UV-A light source 
UV-A LED 
365 nm 
≤40 mW/cm2 
 
Environmental Conditions 
 
IN USE 
STORAGE 
TRANSPORT 
Temperature 
+15 +30° C 
-20 +60° C 
-20 +60° C 
Relative humidity 
25 - 75%  
(non condensing) 
10 - 80% 
(non condensing) 
10 - 80%  
(non condensing) 
Atmospheric pressure 
800 - 1060 hPa 
800 - 1060 hPa 
(-) 
 

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Electrical Data 
Power supply 
AC 100-240V  50/60 Hz 
Power consumption 
<100 VA 
Fuse  
Type 
20 x 5 mm 
Value 
T 2.5 A L 250 V anti-surge 
 
On-Board PC Specifications 
Operating system  
Linux 
Processor 
Intel® Celeron® Processor N3350 
Intel® ATOM x5 Processor E3940 
RAM 
2x2GB DDR3L RAM 
Hard disk 
SSD 128GB 
External connections 
LAN integrated, 2x USB 2.0 
 
Touch Screen 
Dimension  
10” 
Ratio 
16/10 
Resolution 
Full HD 
Rotation 
Rotation in an horizontal plane of ±180° 
External connections 
Recommended to be used with gloves.   
Gloves are two layers of either non-latex, latex or nitrile. 
 

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System Mechanical Features 
Width (max) 
480 mm (1320 mm) 
Height (max) 
1399 mm (1487 mm) 
Length (max) 
560 mm (1435 mm) 
Weight 
31 kg 
Cart wheels 
4 braked swivel wheels with non -marking thermoplastic rubber  
Degrees of freedom of the balanced arm 
5 
 
Main UV-A medical device operation  
• 
Before any intervention, the C4V CHROMO4VIS must be placed adjacent to the treatment table 
or chair, away from bright lights, such as positioning in front of windows and the casters must 
be locked to secure the device’s position.  
• 
The device power is turned on by the medical doctor (the “user” or “operator”). The device then 
checks for startup errors and if the system is starting up correctly, a device calibration is 
performed. 
• 
The system includes three types of users, such as the Administrator, the Operator (i.e., the 
surgeon) and the Service. Only the Administrator has database-level privilege and can 
add/delete surgeons. Therefore, to access patient database, the Operator must be authorized 
by the Administrator, who has database-level privilege. Once authorized by the Administrator, 
the Operator can access to the operator main panel and set the treatment parameters for the 
cornea to treat and the patient’s information. 
• 
To begin preparing for treatment, the user enters and/or select the treatment settings: 
- 
The induction period for the instillation of the 0.22% riboflavin ophthalmic solution in minutes 
and seconds.  
- 
Type of UV-A illumination (continuous or pulsed) 
- 
UV-A energy dose  
- 
UV-A power irradiance 
- 
Once the UV-A energy dose and irradiance have been set, the system calculates the UV-A 
irradiation treatment time in minutes and seconds.  
• 
The user adds the eye to be treated and the corneal parameters (CCT and Kmax) and confirms 
to begin treatment. 

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• 
The user prepares the eye for treatment.  
• 
The device tracks the induction time and notifies the user that the induction is complete.  
• 
The UV-A treatment is then performed.  
• 
The device tracks the UV-A treatment time, turns off the UV-A and notifies the user when the 
treatment has been completed.  
• 
Once the treatment has been completed, the user may choose to treat another patient or the 
device may be powered off.  
 
UV-A irradiation plans 
The system is pre-programmed with three pre-set treatment plans: 
• 
“Epi Off standard” or conventional CXL (15 minutes riboflavin dosing and 30 minutes UV-A 
irradiation by 3 mW/cm2). This is the gold standard procedure for corneal cross-linking. 
• 
“Epi Off accelerated” CXL (15 minutes riboflavin dosing and 9 minutes UV-A irradiation by 10 
mW/cm2). This is the most described high-irradiance procedure for corneal cross-linking in 
bibliography. 
• 
“Epi On accelerated” CXL (20 minutes riboflavin dosing and 9 minutes UV-A irradiation by 10 
mW/cm2). This is the most described high-irradiance procedure for transepithelial corneal cross-
linking in bibliography. 
• 
Any of the pre-set plans can be edited to change any treatment parameters to create a new pre-
set plan that can be saved by pressing the “SAVE” button for future use. 
• 
Any of the pre-set plans can be renamed and then saved for future use. 
 
UV-A irradiation types 
The Operator can select the following types of Procedures from the drop down menu of Treatment 
“Protocol”: 
• 
Epithelium Off protocol (EpiOff).  
• 
Transepithelial protocol (EpiOn).  
- 
When the Epi On protocol is selected, the option “Enhanced EpiOn” is enabled.  
- 
By selecting the “Enhanced EpiOn” option, the UV Energy Dose is increased by 10%. 
 
NOTE: After selecting the “EpiOn protocol” and enabling the “Enhanced EpiOn”, the UV Power 
Density, the UV Delivery Time and the UV-A Energy Dose are automatically increased by 10%.  
 

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The Operator can select the following modalities of UV Irradiation from the drop down menu of UV 
Irradiation “Mode”: 
• Continuous Mode: the UV output is constant for the duration of the UV treatment. The continuous 
mode parameters include the following:  
- 
UV Power: 3 – 36 mW/cm2 (up to 40 mW/cm2 in “Enhanced EpiOn” protocol) 
- 
UV Irradiation Time: 2 minutes – 30 minutes  
- 
UV Energy: 3.6 J/cm2 – 10.0 J/cm2  
• Pulsed Mode: the UV output turns ON and OFF at 1 second interval. The Pulsed Mode 
Parameters include the following:  
- 
UV Power: 3 – 36 mW/cm2 (up to 40 mW/cm2 “Enhanced EpiOn” protocol) UV Irradiation 
Time: 4 minutes – 24 minutes  
- 
UV ON Time: 1.0 Second  
- 
UV OFF Time: 1.0 Second  
- 
UV Energy: 3.6 J/cm2 – 10.0 J/cm2  
 
NOTE: the UV Energy (“Dose”) is the product of the UV Power (“Irradiance”) and the UV 
Irradiation Time. The UV Energy (“Dose”) and the UV Power (“Irradiance”) are adjustable and the 
calculated UV Irradiation Time is displayed. After entering the UV Energy Dose and UV Power 
Density, the UV Delivery Time is automatically calculated and displayed. 
 
NOTE: the C4V CHROMO4VIS system tracks UV Energy, UV Power, UV Irradiation Time and 
Total Treatment Time during the treatment. These options are selectable by the user during the 
treatment plan mode.  
 
The operator may vary the UV Beam Diameter. Use the arrows to increase or decrease the field and 
enter the desired UV Beam Diameter in the corresponding menu.  
 
Detailed information on the correct use of the C4V CHROMO4VIS system can be found in the User 
manual v. 1.3 – For Research Use Only (Annex 9). 
 
Contraindications 
Conditions that may contraindicate the use of the UV-A medical device include:  
• 
Corneal thickness of less than <370 micrometers.  

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• 
Aphakic patients.  
• 
Pregnant and nursing women.  
• 
Eight years old or younger children. 
In these situations, the device should not be used because the risk of use may outweigh any possible 
benefit. 
 
Adverse Events 
Corneal cross-linking is a treatment aiming to increase the biochemical stability of the corneal tissue, 
which has been weakened by disease or surgery, such as keratoconus and corneal ectasia following 
laser vision correction surgery. The gold standard corneal cross-linking procedure requires the 
corneal epithelial removal prior to application of a riboflavin ophthalmic solution and subsequent UV-
A irradiation of the cornea. The standard procedure has been associated with rare severe adverse 
events, including infectious keratitis, anterior uveitis and corneal scars (overall incidence <0.2%), 
which may cause loss of two or more Snellen lines of corrected visual acuity. More recent corneal 
cross-linking procedures, which do not require for epithelial removal, have not been yet associated 
with permanent adverse events of the cornea or eye. The appropriate application of a photo-
protective ophthalmic solution prior to UV-A irradiation of the cornea is a fundamental pre-
requisite to ensure treatment safety. 
The operator shall ensure to follow all instructions in this manual in order to minimize any possible 
risk associated with the use of the C4V CHROMO4VIS system and corneal cross-linking procedures.  
 
Precautions 
• 
Contact lens use must be discontinued for at least 3 days before undergoing corneal cross-
linking. 
• 
Medical gloves are recommended to be worn. 
• 
Only the patient’s eye that is to be treated should be exposed to the UV radiation.  
• 
Physicians should evaluate the potential benefits in patients with the following conditions:  
- 
herpes simplex, herpes zoster keratitis;  
- 
recurrent corneal erosion;  
- 
epithelial healing disorders;  
- 
pseudophakic patients. 
 
 

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List of International Standards 
The C4V CHROMO4VIS™ system conforms to the followings standards: 
• 
UNI CEI EN ISO 15223-1: 2017. Symbols to be used in medical device labels, labeling and 
information to be provided - Part 1: General requirements. 
• 
EN 1041: 2008- Information supplied by the manufacturer with medical devices. 
• 
UNI CEI EN ISO 14971: 2012 - Medical devices - Application of risk management to medical 
devices. 
• 
Decree of the Ministry of Health 15/11/2005. 
• 
Decrees of the Ministry of Health dated 20/03/07 and 21/12/09. 
• 
MEDDEV. 2.7.1 Rev. 4 of June 2016 - Guidelines on medical devices, clinical evaluation: a 
guide for manufacturers and notified bodies. 
• 
MEDDEV 2.4 / 1 Rev. 9 of June 2010 - Guidelines Relating To The Application Of The Council 
Directive 93/42 / EEC on Medical Devices. 
• 
MEDDEV 2.12 -2 Rev. 2 of 11 June 2020 - Guideline on the system of supervision of medical 
devices. 
• 
NB-MED / 2.12 / Rec1. Post-Marketing Surveillance (PMS) post market / production. 
• 
MEDDEV 2.12-2 Rev. 2 January 2012 - Post Market Clinical Follow-Up Studies. 
• 
CEI EN 60601-1 / A1: 2014 Variant - Electro-medical equipment. Part 1: General requirements 
relating to basic safety and essential performance. 
• 
CEI EN 60601-1-2: 2018 - Electromedical equipment. Part 1: General requirements relating to 
basic safety for essential performance - Collateral standard: electromagnetic compatibility - 
Requirements and test. 
• 
CEI EN 60601-1-6: 2011 - Electromedical equipment - Part 1: General requirements relating to 
basic safety and essential performance - Collateral standard: Usability. 
• 
CEI EN 62304: 2015 - Software for medical devices - Processes related to the software life cycle 
(harmonized standard pursuant to Commission Implementing Decision (EU) 2020/437 of 24 
March 2020 is EN 62304: 2006 which represents the technological progress of the standard 
itself and the practical solutions for the management and documentation of the project). 
• 
EN 62366: 2008 + A1: 2015 - Medical devices Application of usability engineering to medical 
devices (harmonized standard pursuant to Commission Implementing Decision (EU) 2020/437 
of 24 March 2020 is EN 62366: 2008 without A1: 2015 which represents the technological 
progress of the standard itself with improvements in relation to the process of usability 
engineering (HUMAN FACTORS ENGINEERING) which allows the company to assess and 

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mitigate the risks associated with the use of the devices in normal operation and in the event of 
abnormal use). 
• 
UNI EN ISO 10993-1: 2010 - Biological evaluation of medical devices - Part 1: Evaluation and 
tests within a risk management process. 
• 
Legislative Decree no. 49/2014 (WEEE) transposition of Directive 2012/19 / EU DIRECTIVE 
2011/65/EU ROHS II of 8 June 2011 on the restriction of the use of dangerous substances in 
electrical and electronic equipment. 
• 
UNI EN ISO 15004-1: 2009 Ophthalmic instruments - Fundamental requirements and test 
methods - Part 1: General requirements applicable to all ophthalmic instruments. 
• 
EN ISO 15004-2: 2007 Ophthalmic instruments - Basic requirements and test methods - Part 2: 
Protection against hazards due to light (non-harmonized standard under the Commission 
Implementing Decision (EU) 2020/437 of 24 March 2020 is the descriptive part of the ISO 15004-
1 test methodology). 
• 
CEI EN 62471: 2010 - Photobiological safety of lamps and lamp systems. (non-harmonized 
standard pursuant to Commission Implementing Decision (EU) 2020/437 of 24 March 2020 
which represents the state of the art for the definition of photo-biological safety of lamps and 
lamp systems, therefore applicable to the device). 
 
 
 
 
 

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Assessing the theranostic software module 2.0 
Theranostics is a cutting-edge treatment paradigm. The term “theranostics” refers to the 
simultaneous integration of therapy and diagnostics and designates a medical solution that provides 
a precise and personalized therapy for the patient. In this specific case, the medical device C4V 
CHROMO4VIS 2.0 enables the surgeon to perform the corneal cross-linking procedure by analyzing 
the images of the corneal tissue to treat. 
The C4V CHROMO4VIS 2.0 system provides estimates of the riboflavin concentration in the cornea 
in real time during treatment, i.e., the riboflavin score, and a predictive index of therapeutic efficacy, 
i.e. the theranostic score. The purpose of this clinical investigation is to validate the theranostics 
software v. 2.0 of the medical device in order to determine the threshold of the theranostic imaging 
biomarker predicting efficacy of the corneal cross-linking procedure, regardless of the CXL treatment 
protocol. 
 
Theranostic-guided UV-A irradiation plans 
For the purpose of this clinical study, the system is pre-programmed with two specific pre-set 
treatment plans: 
• 
“Theranostic EpiOff CXL” consisting of 15 minutes riboflavin dosing and 9 minutes UV-A 
irradiation by 10 mW/cm2.  
• 
“Theranostic EpiOn CXL” consisting of 20 minutes riboflavin dosing and 9 minutes UV-A 
irradiation by 10 mW/cm2.  
 
Theranostic-guided CXL operation sequence 
• 
The device power is turned on by the research medical doctor (the “user” or “operator”). The 
device then checks for startup errors and if the system is starting up correctly, a device 
calibration is performed. 
• 
The research medical doctor with Administrator user privilege can authorize the Operator user 
to access patient database and the operator main panel to set the treatment plan for the cornea 
to treat and the patient’s information. 
• 
Patient data include the key code of the eligible patient to be treated. 
• 
In the treatment plan screen, the Operator adds the eye to be treated and the corneal 
parameters (CCT and Kmax) and confirm to begin treatment. 
• 
The Operator prepares the eye for treatment.  
• 
Once the Operator has focused the system onto the eye, a baseline image of the cornea must 
be acquired by pressing the foot switch for subsequent theranostic real time image analysis. 

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• 
During the application phase, apply one drop of riboflavin ophthalmic solution every 20 second 
directly onto the cornea of the eye to treat. 
• 
During the application phase, the system tracks the application time of riboflavin and notifies the 
user that the induction is complete. The system provides the Operator with an index estimating 
the concentration of riboflavin into the cornea.  
- 
EpiOFF protocol: before measuring the riboflavin concentration into the cornea, dry 
the corneal surface with a tnt stick. 
- 
EpiON protocol: before measuring the riboflavin concentration into the cornea, rinse 
the corneal surface with balanced salt solution. 
• 
The UV-A treatment is then performed. The device tracks the UV-A treatment time, turns off the 
UV-A and notifies the user when the treatment has been completed. The system provides the 
Operator with an index estimating the treatment efficacy, i.e., the theranostic imaging biomarker. 
- 
The operator may choose to rinse the corneal surface with balanced salt solution at 
5 minutes interval during UV-A irradiation. 
• 
Once the treatment has been completed, the Operator save the treatment report in a USB stick 
for printing and adding it to the medical patient record.  
• 
The Operator may choose to treat another eligible patient or the device may be powered off.  
 
 
 

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Pre-clinical studies on safety of the UV-A medical device 
The C4V CHROMO4VIS system has been designed and built in full compliance with EU Directive 
93/42/EEC. The medical device has undergone safety testing according to the following standards: 
• 
EMC EN 60601-1-2 
• 
EN 60601-1  
• 
ISO 15004-2:2007  
• 
EN 64271:2010 
According to the safety testing reports (“R-EM-1435-0920-01A" and “R-EL-1435-0920-03A” by 
Eletrra srl and “3F-RT20006BIS” by CNR-INO), the C4V CHROMO4VIS conforms to the following 
harmonized standards: 
• 
According to EN 60601-1 medical device electrical standard, the C4V Chromo4Vis device is 
classified as:  
o Protection against electrical shock: Class I (external electrical power source).  
o Degree of protection against electric shock: Not classified, equipment not provided with 
applied part.  
o Protection indirect contacts: Class I.  
o Compliance level for protection: IP20 (EN 60529). 
o Method of sterilization or disinfection: disinfectable device.  
o Fire protection: Enclosure has strength and rigidity necessary to prevent a fire  caused 
by reasonably foreseeable misuse.  
o Mode of operation: EUT classified for continuous operation. 
• 
According to FCC Part 15, EN55011 and EN60601-1-2 (electromagnetic emissions): 
o Class A, Group 1. 
o The C4V CHROMO4VIS device can be used in all medical environments (clinics, 
doctors' offices, hospitals etc.) and can be connected directly to a low voltage public 
mains power supply that powers buildings used for commercial or hospital purposes. 
o The C4V CHROMO4VIS device has been tested under different conditions and its 
essential performance has been maintained.  
 
 
 
 
 
 

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Results of the emission and immunity testing  
Port 
Frequency 
range (MHz) 
Test level 
Test 
Reference 
standard 
Outcome 
AC power 
supply 
- 
- 
Harmonic current 
emissions 
EN 61000-3-2 
Ok 
AC power 
supply 
- 
- 
Flicker 
EN 61000-3-3 
Ok 
AC power 
supply 
0.15 – 30 
Class A, Group 1 
Conducted emission 
EN 55011 
Ok 
Enclosure 
30 – 1000 
Class A, Group 1 
Radiated emission 
EN 55011 
Ok 
Enclosure 
1000 – 6000 
Class A, Group 1 
Radiated emission 
EN 55011 
Ok 
Enclosure 
1000 - 18000 
Class A, Group 1 
Radiated emission 
EN 55011 
Ok 
AC power 
supply 
- 
±2KV air 
Immunity against 
fast transients 
(Burst) 
EN 61000-4-4 
Ok 
AC power 
supply 
- 
Common mode: 
±0.5KV; ±1KV; 
±2KV 
Immunity against 
slow transients 
(Surge) 
EN 61000-4-5 
Ok 
AC power 
supply 
- 
Differential 
mode: ±0.5KV; 
±1KV; ±2KV 
Immunity against 
slow transients 
(Surge) 
EN 61000-4-5 
Ok 
Enclosure 
- 
30A/m 
Power frequency 
magnetic fields 
immunity test 
EN 61000-4-8 
Ok 
AC power 
supply 
- 
0%Vn – 10 ms 
0%Vn – 20 ms 
70%Vn – 500 ms 
0%Vn – 5000 ms 
Immunity to dips 
and voltage 
variations 
EN 61000-4-11 
Ok 
Enclosure 
- 
±8KV Contact 
±2KV Air 
±4KV Air 
±8KV Air 
±15KV Air 
±8KV Indirect 
Immunity to 
discharge 
EN 61000-4-2 
Ok 
Enclosure 
80-2700 
3V/m 
Modulated AM 
80% 1KHz 
Radiated 
electromagnetic 
field immunity test 
EN 61000-4-3  
EN 60601-1-2  
Ok 

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Enclosure 
704-5800 
9V/m 
Modulated PM 
50% 217Hz 
Radiated 
electromagnetic 
field immunity test 
EN 61000-4-3  
EN 60601-1-2  
Ok 
Enclosure 
380-390 
27V/m 
Modulated PM 
50% 18Hz 
Radiated 
electromagnetic 
field immunity test 
EN 61000-4-3  
EN 60601-1-2  
Ok 
Enclosure 
430-960 
28V/m 
Modulated PM 
50% 18Hz 
Radiated 
electromagnetic 
field immunity test 
EN 61000-4-3  
EN 60601-1-2  
Ok 
Enclosure 
1700-2570 
28V/m 
Modulated PM 
50% 217Hz 
Radiated 
electromagnetic 
field immunity test 
EN 61000-4-3  
EN 60601-1-2  
Ok 
 
• 
According to EN ISO 15004-2:2007 Ophthalmic instruments – Fundamental requirements 
and test methods – Part 2: Light hazard protection: 
o Blue LED is Risk Group 1. 
o Green LED is Risk Group 1. 
• 
According to EN 62471:2010 Photobiological safety of lamps and lamp systems: 
o UVA LED is Risk Group 3. 
• 
According to rule 9 of Annex IX of Directive 93/42/EEC and MEDDEV 2.4/1. Rev.9: 
o Class IIb. 
 
 

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Pre-clinical studies on efficacy of the theranostic UV-A medical device 
The efficacy and performance of the UV-A medical device in question has been validated in the 
laboratory on donor eye bank corneal tissues. The laboratory experiments on the UV-A medical 
device under evaluation were performed in accordance with the principles of the Helsinki Declaration 
for the use of human tissues in scientific research. The donor corneal tissues were shipped to the 
manufacturer’s laboratory by the Fondazione Banca degli Occhi del Veneto (Venezia Zelarino) after 
adequate time of cultivation in special media in order to preserve the integrity of the corneal 
epithelium and the tissue integrity and transparency. 
In the first pre-clinical study, 6 a former group of seven (7) corneal tissues was treated by using the 
Dresden protocol, i.e., with application of riboflavin for 30 minutes and UV-A irradiance of 3 mW/cm2 
for 30 minutes (total 5.4 J/cm2 energy dose). An additional group of ten (10) corneal tissues was 
probed by atomic force microscopy (AFM) in order to assess the biomechanical properties of the 
cornea after corneal cross-linking using the Dresden protocol. The scope of the study was to 
evaluate the performance of the present device in improving the corneal biomechanics strength, 
measured as increased tissue stiffness (i.e., increased Young’s modulus). After corneal cross-
linking, the average Young's modulus increased significantly from 1.61±0.53 MPa preoperatively to 
2.08±0.72 MPa postoperatively (P=0.001). In summary, the results of the laboratory validation have 
shown that the UV-A medical device in question is effective in performing corneal cross-linking 
treatment on human eye corneal tissues by increasing the elastic modulus of the corneal tissue. No 
alteration of the corneal tissues undergoing corneal cross-linking with the UV-A medical device in 
question was observed. 
In the second pre-clinical study,7 fourteen (14) eye bank donor corneal tissues were divided into two 
study groups and treated by the UV-A medical device under evaluation after appropriate application 
of riboflavin onto the cornea. The first group of seven (7) corneal tissues underwent corneal cross-
linking with the Dresden protocol (i.e., application of riboflavin for 30 minutes after de-epithelialization 
and UV-A irradiance of 3 mW/cm2 for 30 minutes with total 5.4 J/cm2 energy dose); the second study 
group of 7 corneal tissues underwent the accelerated corneal cross-linking protocol by using 10 
mW/cm2 UV-A power density for 9 minutes (total 5.4 J/cm2 energy dose). Pre-irradiation corneal 
soaking for 30 minutes achieved highly consistent values of intrastromal riboflavin concentration 
(0.015%±0.003%) between tissues. After UV-A irradiation of the cornea, the mean intrastromal 
riboflavin concentration was 0.003%±0.001%, with no significant difference between the two UV-A 
irradiation protocols (P=0.40). In summary, the study showed that the two treatment protocols 
 
6 Lombardo G et al. Non-invasive optical method for real-time assessment of intracorneal riboflavin concentration and efficacy of corneal cross-linking. 
J Biophotonics 2018; Jul;11(7):e201800028. 
7 Lombardo M, Lombardo G. Non-invasive and real time assessment of riboflavin consumption in standard and accelerated corneal cross-linking. J 
Cataract Refract Surg 2019; 45(1):80-86. 

ARGO - ver. 1.0 28/06/2021 
 
 
 
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performed with the UV-A medical device in question on human eye bank corneal tissues were 
equivalent in terms of efficacy and performance. No alteration of the corneal tissues undergoing 
corneal cross-linking with the UV-A medical device in question was observed. 
In the third pre-clinical study,8 ten (10) eye bank corneal tissues were divided into two study groups 
and treated by the UV-A medical device in question after appropriate application of riboflavin onto 
the cornea. The first group of five (5) corneal tissues underwent corneal cross-linking with the 
Dresden protocol (i.e., application of riboflavin for 30 minutes onto the cornea after de-
epithelialization and UV-A irradiance of 3 mW/cm2 for 30 minutes with total 5.4 J/cm2 energy dose); 
the second study group of 5 corneal tissues underwent transepithelial corneal cross-linking with 
iontophoresis protocol with epithelium intact (i.e., application of riboflavin for 5 minutes at 1mA/min 
with corneal iontophoresis followed by UV-A irradiance of 10 mW/cm2 for 9 minutes with and total 
5.4 J/cm2 energy dose). The application of riboflavin for 30 minutes onto the stroma achieved high 
corneal riboflavin concentration values (425±77 µg/cm3); during UV-A irradiation, the stromal 
riboflavin concentration decreased non linearly in all tissues; at the end of irradiation period, the 
mean intrastromal riboflavin concentration was 54±29 µg/cm3, i.e., on average 87% lower than the 
concentration value obtained at the end of soaking phase. After corneal iontophoresis with intact 
epithelium, the mean corneal concentration of riboflavin was 195±35 µg/cm3 (values significantly 
lower, P=0.001, than conventional corneal soaking); at the end of irradiation period, the mean 
riboflavin concentration was 31±9 µg/cm3, i.e., on average 85% lower than the concentration value 
achieved at the end of soaking phase. In summary, the study showed that UV-A medical device 
under evaluation can perform either treatment protocols efficiently and that the results of the Dresden 
protocol are superior compared to those of the transepithelial protocol. No alteration of the corneal 
tissues undergoing corneal cross-linking with the UV-A medical device in question was observed. 
In conclusion, the laboratory studies on the UV-A medical device under evaluation have provided 
sufficient data and information on the safety and performance of the device for the intended purpose 
of use in an adequate number of human corneal samples. Such data are sufficient to verify that the 
UV-A medical device is in conformity with all the Essential Requirements pertaining to clinical 
performance and clinical safety. 
 
 
 
8 Lombardo G, Serrao S, Lombardo M. Comparison between standard and transepithelial corneal cross-linking using a theranostic UV-A device. 
Graefe’s Arch Clin Exp Ophthalmol 2020; 5 Gen E-pub. 

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Sterile ophthalmic riboflavin solution 
Product Description 
Ritsight™ is an ophthalmic solution containing riboflavin (0.22%), formulated to allow the penetration 
of riboflavin into the corneal stroma before ultraviolet (UV-A) irradiation during corneal cross-linking 
procedures. The purpose of Ritsight is to soak the corneal stroma with riboflavin to constitute a 
protective barrier against the penetration of UV-A rays beyond the corneal stroma, preserving the 
internal structures of the eye (corneal endothelium, lens and retina) from damage that light radiation 
could cause them during corneal cross-linking procedures. 
 
Indications for use 
Ritsight™ is indicated to protect the internal ocular structures from excessive exposure to UV-A light 
rays in the treatment of ectatic corneal diseases, such as keratoconus and iatrogenic corneal ectasia, 
during corneal cross-linking procedures. 
 
Mode of use 
At the beginning of the imbibition phase of corneal cross-linking, Ritsight shall be applied directly 
onto the cornea, pressing the bottle with the fingers, until the entire corneal surface is wet. Therefore, 
a drop of Ritsight shall be applied onto the cornea 3 times per minute for at least 15 minutes before 
UV-A irradiation. Once the soaking phase is complete, proceed with UV-A irradiation of the cornea 
according to the specific corneal cross-linking procedure. The emitted UV-A irradiation power or the 
UV-A energy dose must not exceed 40 mW/cm2 or 10 J/cm2 respectively. It is recommended to use 
UV-A emitters capable of verifying the emitted UV-A energy or the concentration of riboflavin on the 
corneal surface. 
 
Composition 
Riboflavin sodium phosphate, sodium dihydrogen phosphate dihydrate, disodium phosphate 
dodecahydrate, sodium chloride, water for injection. 
 
Description main ingredient 
Composition 
Riboflavin sodium phosphate 
0.30% (equivalent to 0.22% free base riboflavin) 
 
Contraindication 
The product is contraindicated in case of known hypersensitivity to components or other substances 
closely related from a chemical standpoint. 

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Side effects 
Following the instillation of riboflavin ophthalmic solutions, there are no known systemic or local side 
effects on the ocular structures. Cross-linking treatment is associated with rare severe adverse 
events, including infectious keratitis, corneal burn and corneal leucoma, the cumulative incidence of 
which is lower than 0.8%. The incorrect execution of cross-linking treatment, such as insufficient 
application of riboflavin on the cornea, can increase the risk of onset of side effects, including 
phototoxic reactions and infections as well as can make the treatment ineffective. 
 
Precautions 
• 
Do not use the product after the expiry date indicated on the package. 
• 
Do not use if the bottle is damaged. 
• 
The medical device must be used immediately after its first opening. 
• 
Each bottle must be used for one treatment only. Any use for multiple treatments may lead 
to the onset of eye infections. 
• 
Do not use fluorescein on the cornea before instilling the product. 
• 
The medical device is for the exclusive use of the ophthalmologist or under his/her direct 
control. 
• 
Do not use the medical device outside of the indications for use. 
• 
Do not use in pregnant or nursing women. 
• 
Do not use in 8 years or younger children. 
• 
Store the product away from light and at temperatures between 4° C and 25° C. 
• 
After its first use, the medical device must not be stored but must be disposed of as hospital 
waste or according to current regulations. 
 
 
 

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Pre-clinical studies on safety of the 0.22% riboflavin ophthalmic solution 
The 0.22% riboflavin ophthalmic solution has undergone biocompatibility testing before CE marking, 
including: 
• 
Citotoxicity by direct contact according ISO 10993-5:2009. 
• 
Acute Ocular irritation test according ISO 10993-10:2010.  
• 
Delayed hypersensitivity test according ISO 10993-10:2010. 
The results of the citotoxicity by direct contact (Report STULV20AA3833-1 GLP; Eurofins Biolab srl) 
performed to a monolayer BALB/3T3 cells using the Neutral Red Uptake method (NRU), can be 
summarized as follows: 
Cells treated have shown a cell viability reduction of 5%. 
On the basis of the results, the product must be considered NOT CYTOTOXIC. 
The results of the acute ocular irritation test (Report FR STULV20AA3840-1 GLP; Eurofins Biolab 
srl) performed on three albino female rabbits, can be summarized as follows: 
Cornea: 
• 
Degree of opacity: no abnormalities were observed. 
• 
Area of cornea involved: no abnormalities were observed. 
Iris: no abnormalities were observed. 
Conjunctiva: 
• 
Redness: no abnormalities were observed. 
• 
Chemosis: no abnormalities were observed. 
• 
Discharge: no abnormalities were observed.  
On the basis of the results, the product must be considered NOT IRRITANT at ocular level. 
The results of the delayed hypersensitivity test (Report N° STULV20AA3835-1 GLP; Eurofins Biolab 
srl) performed on fifteen guinea pigs, can be summarized as follows: 
• 
no abnormalities were observed. 
On the basis of the results, the product must be considered NOT SENSITIZING. 
 
 

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Study Plan 
Aim 
The scope of the study is to evaluate a novel modality of riboflavin/UV-A corneal cross-linking based 
on theranostics aiming at improving treatment predictability for better eye care to patients suffering 
from keratocous. The objective of the study is to validate the combined use of theranostic imaging 
biomarker scores in predicting CXL treatment efficacy by assessing the change of corneal 
topography Kmax value at 12 months after riboflavin/UV-A corneal cross-linking for the treatment of 
keratoconus.  
 
Type of study and duration 
This is a multi-centre clinical study consisting of one study arm with two stratified groups to assess 
the performance of a new software version (2.0.) of a CE marked class IIb UV-A medical device, 
C4V CHROMO4VIS (CE1936) for treating keratoconus with corneal cross-linking procedure. 
In this study, patients with progressive keratoconus receives a single dose of the ophthalmic solution 
RitSight™ (CE0477) delivered into the cornea manually for at least 15 minutes and treated by using 
the C4V CHROMO4VIS™ system with the theranostic software module while they are observed and 
tested for a period of 12 months after treatment to assess correlation between the theranostic 
imaging biomarker and the Kmax flattening.  
Follow-up for each participant is twelve months after treatment. The interval is long enough to 
observe treatment efficacy as well as any effect or complication induced by the innovative corneal 
cross-linking procedure performed by using the theranostic UV-A medical device in a participant. 
 
Study Participants 
The inclusion criteria are: diagnosis of progressive keratoconus. Participants with 18-40 years old 
are eligible, all genders. 
According to the most recent scientific literature9, the criterion to determine progression of 
keratoconus is based on providing at least one of the following types of evidence:  
• 
at least two Placido disk corneal topography measurements showing at least +1.00 D 
steepening of the Kmax value in the last year or longer interval period. 
• 
at least two central corneal thickness (CCT) measurements showing at least -10 µm change 
in in the last year or longer interval period. 
 
9 Ferdi A, et al. Keratoconus Natural Progression: A Systematic Review and Meta-analysis of 11 529 Eyes. Ophthalmology 2019;126:935-945 

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The exclusion criteria are: corneal apex steeper than 63 D, corneal thickness thinner than 400 µm; 
corneal scarring; descemetocele; history of herpetic keratitis; concomitant eye diseases; 
inflammatory eye diseases; glaucoma; cataract; nistagmus; pregnancy; breast feeding. 
The study does not accept healthy volunteers. 
If both eyes of a participant are eligible, the eye with lower CDVA will be chosen as the study eye. 
 
Study Arm 
One study arm receiving either corneal cross-linking treatment after epithelial debridement 
(“Theranostic Epi-OFF CXL”) or transepithelial corneal cross-linking treatment (“Theranostic Epi-ON 
CXL”). Only one eye of each participant is designated as the study eye. 
 
Discontinuation of contact lens wear 
Participants must discontinue the use of contact lenses from seven days before baseline visit to 3 
months follow-up; thereafter, they must discontinue the use of contact lenses at least seven days 
before the remaining follow-up visits at day-180 and day-360. 
 
Outcome Measures and Assessments 
Both eyes of each participant will be evaluated at baseline (i.e., before treatment) to assess eligibility; 
only one eye for each participant will be treated by corneal cross-linking (“study eye”). The study eye 
of each recruited subject will be assessed to investigate the primary and secondary outcome 
measures. 
Assessment of the novel theranostic-guided riboflavin/UV-A corneal cross-linking treatment modality 
will be determined objectively by a comprehensive eye examination, measurement of corneal 
topography and corneal endothelial microscopy. 
Follow-up assessments will be performed on day 7, 30, 90, 180 and 360 after treatment. 
At baseline (assessment of both eyes) and all the subsequent follow-up visits (assessment of “study 
eye” only) each subject recruited in the study will undergo the following examinations: 
1) Uncorrected Distance Visual Acuity (UDVA) tested using the ETDRS chart. 
2) Corrected Distance Visual Acuity (CDVA) tested using the ETDRS chart. 
3) Manifest Spherical Equivalent Refraction (MSER) tested with ETDRS chart. 
4) Measurement of corneal curvature (Kmax, Ksteep, Kflat) and corneal thickness (CCT) using a 
combined Placido-disk computerized corneal topography. 
5) Measurement of endothelial cell density (ECD) using the corneal specular microscopy. 
6) Slit lamp bio-microscopy of the ocular surface and the anterior segment of the eye.  

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At baseline (assessment of both eyes) and at last follow-up visit (12-months), each subject recruited 
in the study will undergo also ocular tonometry and indirect ophthalmoscopy.  
The study visits and the battery of tests to be performed at each visit are fully described in next 
section. 
The investigators may opt to perform additional, no-contact, instrumental testing, as a part of their 
standard of care practice in patients with keratoconus, which are not due for protocol. A rationale for 
performing additional instrumental testing in participants shall be added in the corresponding box of 
the CRF (Annex 1). 
 
Study Visits  
Each participant will undergo treatment and six study visits. A tabulated summary of the study visits 
for study groups including the assessments to be performed at each visit is summarized below: 
Time 
baseline 
day 0 
day 7 
day 30 
day 90 
day 180 
day 360 
 
 
 
 
 
 
 
 
Description 
Visit 1 
Treatment 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Informed consent 
X 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
UDVA 
X 
 
X 
X 
X 
X 
X 
CDVA 
X 
 
X 
X 
X 
X 
X 
Manifest Refraction 
X 
 
X 
X 
X 
X 
X 
Corneal Topography/Pachymetry 
X 
 
X 
X 
X 
X 
X 
Corneal Specular Microscopy 
X 
 
X 
X 
X 
X 
X 
Slit Lamp Microscopy 
X 
 
X 
X 
X 
X 
X 
Ocular Tonometry  
X 
 
 
 
 
 
X 
Dilated fundus Examination 
X 
 
 
 
 
 
X 
Theranostic data 
 
X 
 
 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
 
Visit 1 – Baseline  
Potential candidates will undergo a complete eye examination to determine their eligibility to the 
study. Before any test is conducted, the participants will be given a brief explanation of the study 
(including the number and time of visits to be performed).  
The baseline visit will provide the confirmation that the participant is eligible for the inclusion in the 
study and the full medical history will be collected. Baseline measurements will be performed in the 
following order:  

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1. Corneal curvature and corneal thickness measurements using the computerized corneal 
topography device. 
2. Endothelial cell density measurement using the specular microscopy. 
3. Uncorrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
4. Corrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
5. Manifest Spherical Equivalent Refraction with ETDRS chart at a test distance of 4 m. 
6. Slit lamp bio-microscopy of the ocular surface and the anterior segment of the eye. 
7. Intra-ocular pressure (IOP) measurement. 
8. Dilated fundus examination. 
At the end of the baseline assessment, eligible participants shall the informed consent form (Annex 
3) and will be assigned an individual stratification number according to the stratification protocol 
described in the corresponding section of this study protocol.  
Participants will be also reminded to bring sunglasses the day of surgery. 
 
Treatment (day 0) 
Participants will receive theranostic-guided cross-linking treatment according to the RUO User 
Manual and the Investigator’s Brochure instructions and the following section “Treatment”.  
After treatment, each participant will receive medical therapy and relative instructions (Annex 3). 
 
NOTE: To provide standard medical care, the participants treated by EpiOFF theranostic-
guided CXL will be visited at day 3 to discontinue the use of contact lens in the treated eye. 
 
Visit 2, 3, 4 and 5 – Follow up (day 7, day 30, day 90 and day 180 respectively) 
These visits will provide follow-up measurements, which will be performed in the following order:  
1. Corneal curvature and corneal thickness measurements using the computerized corneal 
topography device. 
2. Endothelial cell density measurements using the specular microscopy. 
3. Uncorrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
4. Corrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
5. Manifest Spherical Equivalent Refraction with ETDRS chart at a test distance of 4 m. 
6. Slit lamp bio-microscopy of the ocular surface and the anterior segment of the eye. 
Any adverse event occurring between the previous and the current visit will be recorded. 

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A ±2 working day tolerance window will be allowed for visit 2; a ±4 days tolerance window will be 
allowed for visit 3; a ±10 days tolerance window will be allowed for visit 4 and a ±14 days tolerance 
window will be allowed for visit 5. 
 
Visit 6 – Follow up (day 360) 
This visit will provide last follow-up measurements, which will be performed in the following order:  
1. Corneal curvature and corneal thickness measurements using the computerized corneal 
topography device. 
2. Endothelial cell density measurements using the specular microscopy. 
3. Uncorrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
4. Corrected Distance Visual Acuity, measured with ETDRS chart at a test distance of 4 m. 
5. Manifest Spherical Equivalent Refraction with ETDRS chart at a test distance of 4 m. 
6. Slit lamp bio-microscopy of the ocular surface and the anterior segment of the eye. 
7. Intra-ocular pressure (IOP) measurement. 
8. Dilated fundus examination. 
Any adverse event occurring between the previous and the current visit will be recorded. 
A ±21 working days tolerance window will be allowed for visit 6, which will be the last study visit for 
all participants enrolled in the study. 
 
Dropouts 
Participants may drop out at any time during the study. The list of drop out (Annex 4) shall include 
information on participants who drop out from the study. 
 
 
 

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Treatment 
In this clinical study, the innovative corneal cross-linking procedure will be performed using the 
theranostic software module of the C4V CHROMO4VIS™ system in all participants. Participants will 
receive a single dose of the 0.22% riboflavin ophthalmic solution, RitSight™. Application of the 
riboflavin eye drop will be done for at least 15 minutes in each participant, specifically 15 minutes for 
the EpiOFF CXL treatment and 20 minutes for the EpiON CXL treatment. Estimates of riboflavin 
concentration into the cornea will be monitored by the C4V CHROMO4VIS™ system during the 
dosing phase of treatment. Upon reaching a minimum threshold concentration into the cornea, 
defined by the C4V CHROMO4VIS™ system, the system provides the Operator the access to the 
UV-A irradiation of the cornea with 5.4 J/cm2 total energy dose and 7.00 mm light beam diameter in 
all participants. 
Patient data: in order to anonymize patient data, the user shall code name using the stratification 
code provided by the Sponsor at the time of enrolment. 
Treatment settings are pre-set for both treatment protocols that are object of investigation in the 
present clinical study: 
Investigational theranostic-guided  
EpiOFF protocol 
Investigational theranostic-guided  
EpiON protocol 
- 
Riboflavin dosing mode: manual 
- 
Riboflavin dosing time: 15 minutes 
- 
UV-A irradiation mode: continuous. 
- 
UV-A irradiation power: 10 mW/cm2 
- 
UV-A irradiation time: 9 min. 
- 
UV-A energy dose: 5.4 J/cm2 
- 
Beam aperture: 7 mm 
- 
Riboflavin dosing mode: manual 
- 
Riboflavin dosing time: 20 minutes 
- 
UV-A irradiation mode: continuous. 
- 
UV-A irradiation power: 10 mW/cm2 
- 
UV-A irradiation time: 9 min. 
- 
UV-A energy dose: 5.4 J/cm2 
- 
Beam aperture: 7 mm 
 
The riboflavin/UV-A corneal cross-linking protocol that shall be used in this study is described as 
follows: 
• 
the C4V CHROMO4VIS™ system must be checked by the user before each use; 
• 
the C4V CHROMO4VIS™ system power is turned on by the user. The device then checks for 
startup errors and if the system is starting up correctly, a device calibration is performed; 
• 
to begin preparing for treatment, the user select the treatment settings: 
- 
Induction period for the instillation of the ophthalmic solution RitSight: manual. 
- 
Type of UV-A illumination: continuous.  
- 
UV-A energy dose: 5.4 J/cm2. 

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- 
UV-A power irradiance: 10 mW/cm2. 
- 
Once the UV-A energy dose and irradiance have been set, the system calculated the UV-
A irradiation treatment time in minutes and seconds; 
• 
the user adds the key code of the eligible patient to be treated in the “patient database screen” 
of the system; 
• 
the user add the eye to be treated and the corneal parameters (CCT and Kmax) in the “treatment 
plan screen” of the system and confirm to begin treatment; 
• 
the user prepares the patient’s eye for treatment (numbing eye drops and eye speculum) and 
access the “treatment screen” of the C4V CHROMO4VIS™ system; 
• 
the user applies the riboflavin ophthalmic solution onto the cornea; the user applies one drop of 
riboflavin every 20 seconds directly onto the cornea of the eye to treat; 
• 
the user must dry (in EpiOFF protocol) or rinse with balanced salt solution (in EpiON protocol) 
the excess of riboflavin onto the cornea before measuring its concentration by pressing the foot 
switch; 
• 
the C4V CHROMO4VIS™ system tracks the riboflavin application time and notifies the user that 
the induction is complete; 
• 
the user selects to proceed to perform UV-A treatment in the “treatment screen” of the C4V 
CHROMO4VIS™ system;  
• 
the user may choose to rinse with a drop of balanced salt solution the corneal surface during 
UV-A irradiation once at 5 minutes interval. 
• 
the C4V CHROMO4VIS™ system tracks the UV-A treatment time, turns off the UV-A and notifies 
the user when the treatment has been completed; 
• 
the user saves the treatment report in an USB stick; 
• 
the user may choose to treat another eligible patient or the C4V CHROMO4VIS™ system may 
be powered off.  
Detailed information on the treatment procedure can be found in the User Manual – For Research 
Use Only (Annex 9), including the pictures of the wizard screens. 
 
Patient Counseling Information  
Patients shall be advised not to rub their eyes for the first week after their procedure.  
Patients may be sensitive to light and have a foreign body sensation. Patients shall be advised that 
there may be discomfort in the treated eye and that sunglasses may help with light sensitivity.  
If patients experience severe pain in the eye or any sudden decrease in their vision, they shall be 
advised to contact the Principal Investigator of the Site of Investigation immediately.  

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If the bandage contact lens that is placed on the patient’s eye on the day of treatment falls out or 
becomes dislodged, the patient shall be advised not to replace it and to contact the Principal 
Investigator of the Site of Investigation immediately.  
 
Study Product Packaging and Storage  
The Site of Investigation will receive an UV-A medical device and enough bottles of the RitSight 
ophthalmic solution to treat all the planned participants. Each Site will receive at least 20 bottles of 
RitSight™. The carton box of each bottle will be labelled with a white printed label bearing the 
following information in English:  
• 
For Clinical Trial Use Only 
• 
Study code: RSKC001 
• 
Acronym: ARGO 
• 
Sponsor: Regensight srl 
 
The participant box shall be stored in a locked cabinet/locked room at the Site of Investigation’s 
operating room between 4°C to 25°C. Only the principal investigator of the Site will have access to 
these products during the course of the study. He/She will annotate the stratification number for that 
participant in the respective CRF. 
 
 Test Article Accountability 
The principal investigator of the Sites of Investigation will maintain accurate records of receipt of all 
study products, including dates of receipt. In addition, accurate records will be kept regarding when 
the study product (participant kit) is dispensed and used by each patient in the study. Reasons for 
departure from the expected dispensing regimen or dispensing of an extra kit must also be recorded. 
At the completion of the study, to satisfy regulatory requirements regarding accountability, all study 
products records will be reconciled. 
 
 

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Study assessments 
Demographic Data/Medical History 
Key-coded personal details, gender, age, details of general health and eye health and current 
medication.  
 
Efficacy Assessments  
Efficacy data will be identified by using objective and subjective methodologies, as described in the 
following: 
• 
Measurement of corneal curvature with Placido disk topographer will assess changes of the 
Kmax after treatment. 
 
Safety Assessments  
Safety data will be identified by using objective methodologies, as described in the following: 
• 
Measurement of Corrected Distance Visual Acuity (CDVA) with ETDRS chart at a test 
distance of 4 m will assess changes of the CDVA after treatment. 
• 
Measurement of corneal endothelial cell density (ECD) with specular microscopy will 
assess will assess changes of the ECD after treatment. 
• 
Slit lamp biomicroscopy will assess integrity of the cornea and anterior segment of the eye. 
 
Primary outcome measure 
Validation of the Theranostic Imaging Biomarker [Time Frame: 12-months] 
• 
Validation of the combined use of the theranostic imaging biomarkers. True positives, false 
positives, true negatives and false negatives were evaluated in order to determine the ability 
of the theranostic imaging biomarkers, including both the riboflavin score and theranostic 
score, to predict the propensity of CXL treatment in flattening the Kmax value (D) by more than 
0.1 D at 12 months postoperatively.  
Theranostics: confirmation of the theranostic imaging biomarker will be provided at the end of 
treatment procedure. The theranostic data, which will be recorded during treatment procedure, will 
be exported to a computer for subsequent analysis with 12-months data. 
The accuracy and precision (95% CI) of the combined use of the theranostic imaging biomarkers to 
predict CXL treatment outcome were determined by calculating the proportion of correctly classified 
eyes and the positive predictive value (PPV) respectively. The miss rate, which is the probability that 
a true positive is missed by the theranostic methodology, was determined by calculating the false 
negative rate (FNR). 

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The study sets a minimum threshold of 85% for the theranostic software module’s accuracy and 
precision in predicting the propensity of CXL to halt disease progression (intended as more than 0.1 
D Kmax flattening) at 1 year in the study population, who participated and completed at least 66% 
of all the visits (i.e., 4 of 6 total visits) and the last at 12 months (i.e., Per Protocol Population; 
 
Secondary outcome measures 
Efficacy [Time Frame: 12-months] 
• 
Change of Maximum Keratometry index assessed by corneal topography at 12-months. 
Corneal topography: measurement of maximum simulated keratometry (Kmax, D) will be done by 
using Placido disk corneal topography. The investigator will record the Kmax value at each visit and 
will fill the corresponding CRF. This test is performed to provide evidence of corneal curvature 
change after treatment. 
 
Safety [Time Frame:  12-months] 
• 
Change of Endothelial Cell Density assessed by specular microscopy at 12-months. 
Corneal specular microscopy: endothelial cell density (ECD, cell/mm2) count will be done by using 
corneal specular microscopy. The investigator will record the ECD at each visit and will fill the 
corresponding CRF. This test is performed to provide evidence of corneal cells integrity after 
treatment. 
 
Other outcome measures 
Efficacy [Time Frame:  12-months] 
• 
Change of Manifest Refraction assessed by ETDRS at 12-months. 
Manifest Refraction: manifest refraction will be determined using a standard ETDRS chart under 
photopic conditions (with the luminance of the test at 85 cd/m2) at a test distance of 4 m. The Manifest 
Refraction will be assessed by using trial lenses and will be expressed in diopters (D). This test is 
performed to provide evidence of optical focusing properties change after treatment. 
• 
Change of Best-Spectacle Corrected Visual Acuity (CDVA) at 12-months. 
Corrected Distance Visual Acuity (CDVA): CDVA will be determined using a standard “Early 
Treatment Diabetic Retinopathy Study” (ETDRS) chart under photopic conditions (with the 
luminance of the test at 85 cd/m2) at a test distance of 4 m. The CDVA will be expressed in LogMAR. 
The ETDRS test incorporates specific design criteria to make it more accurate than the Snellen or 
Sloan acuity tests. These include: 
• 
Same number of letters per row (five letters per row); 

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• 
Equal spacing of the rows on a log scale (the rows are separated by 0.1. log unit); 
• 
Equal spacing of the letters on a log scale; 
• 
Individual rows balanced for letter difficulty. 
The patient starts are the top of the chart and begins to read down the chart. The patient reads down 
the chart until he or she reaches a row where a minimum of three letters on a line cannot be read. 
The patient is scored by how many letters could be correctly identified and by the LogMAR of the 
line with ≥3 letters he/she is able to read. 
• 
Change of Unorrected Visual Acuity (CDVA) at 12-months. 
Uncorrected Distance Visual Acuity (UDVA): UDVA will be determined using a standard “Early 
Treatment Diabetic Retinopathy Study” (ETDRS) chart under photopic conditions (with the 
luminance of the test at 85 cd/m2) at a test distance of 4 m. The UDVA will be expressed in LogMAR. 
The ETDRS test incorporates specific design criteria to make it more accurate than the Snellen or 
Sloan acuity tests. These include: 
• 
Same number of letters per row (five letters per row); 
• 
Equal spacing of the rows on a log scale (the rows are separated by 0.1. log unit); 
• 
Equal spacing of the letters on a log scale; 
• 
Individual rows balanced for letter difficulty. 
The patient starts are the top of the chart and begins to read down the chart. The patient reads down 
the chart until he or she reaches a row where a minimum of three letters on a line cannot be read. 
The patient is scored by how many letters could be correctly identified and by the LogMAR of the 
line with ≥3 letters he/she is able to read. 
Safety [Time Frame:  12-months] 
• 
Change of Central Corneal Thickness assessed by corneal pachymetry at 12-months. 
• 
Corneal pachymetry: measurement of central corneal thickness (CCT, µm) will be done by 
using combined corneal topography/pachymetry device. The investigator will record the CCT 
value at each visit and will fill the corresponding CRF. This test is performed to provide 
evidence of corneal thickness change after treatment. 
 
Adverse Events 
Reporting adverse events 
The Principal Investigator of the Site of Investigation is responsible for reporting all adverse events 
(AEs) that are observed or reported during the study, regardless of their relationship to study 
treatment or their clinical significance.   
An AE is defined as any untoward medical occurrence in a participant enrolled into this study 
regardless of its causal relationship to study treatment. Participants will be instructed to contact the 

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principal investigator of the Site of Investigation at any time if any symptoms develop. A treatment-
emergent AE is defined as any event not present prior to exposure to study product or any event 
already present that worsens in either intensity or frequency following exposure to test medication.  
All AEs that occur during the study must be reported in detail on the CRF and followed to satisfactory 
resolution or until the principal investigator of the Site of Investigation deems the event to be chronic 
or the participant to be stable. The description of the AE will include the onset date, duration, date of 
resolution, severity, seriousness, etiology, and the likelihood of relationship of the AE to study 
treatment.  
A serious adverse event (SAE) is defined as any event that results in death, is immediately life 
threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in 
persistent or significant disability/incapacity, is a congenital anomaly/birth defect. Important medical 
events that may not result in death, be life threatening, or require hospitalization may be considered 
an SAE when, based upon appropriate medical judgment, they may jeopardize the participant and 
may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 
Examples of such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias, or convulsions that do not result in participant 
hospitalization. 
 
Eliciting Adverse Event Information  
Adverse events will be assessed from the time the participant receives the treatment until exit from 
the study.  At every study visit, participants will be asked a standard question to elicit any medically 
related changes in their well-being. They will also be asked if they have been hospitalized, had any 
accidents, used any new medications, or changed concomitant medication regimens (both 
prescription and over-the-counter medications). In addition to participant observations, AEs will be 
documented from any data collected on the AE CRF or other documents that are relevant to 
participant safety. 
 
Adverse Event Reporting  
All AEs reported or observed during the study will be recorded on the AE CRF according to the ICH 
Guidelines. Information to be collected includes dosage, type of event, time of onset, investigator-
specified assessment of severity and relationship to study Treatment, time of resolution of the event, 
seriousness, any required treatment or evaluations, and outcome. Adverse events resulting from 
concurrent illnesses, reactions to concurrent illnesses, reactions to concurrent medications, or 
progression of disease states must also be reported. All AEs will be followed to adequate resolution.  

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Medical Dictionary for Regulatory Activities (MedDRA) will be used to code all AEs. The As will be 
reported in the CRF according to the mdcg 2020-10-2 - Guidance safety report form. 
Any medical condition that is present at the time that the participant is screened but does not 
deteriorate should not be reported as an AE. However, if it deteriorates at any time during the study, 
it should be recorded as an AE. 
 
Assessment of Causality  
The investigator’s assessment of an AE's relationship to study Treatment is part of the 
documentation process, but it is not a factor in determining what is or is not reported in the study.  If 
there is any doubt as to whether a clinical observation is an AE, the event should be reported. 
The relationship or association of the test article in causing or contributing to the AE will be 
characterized using the following classification and criteria: 
Unrelated: 
This relationship suggests that there is no association between the study 
treatment and the reported event. 
Possible:  
This relationship suggests that treatment with the test article caused or 
contributed to the AE, i.e., the event follows a reasonable temporal sequence 
from the time of treatment administration and/or follows a known response 
pattern to the test article, but could also have been produced by other factors. 
Probable:  
This relationship suggests that a reasonable temporal sequence of the event 
with treatment administration exists and, based upon the known action of the 
treatment, known or previously reported adverse reactions to the treatment, or 
judgment based on the investigator’s clinical experience, the association of the 
event with the study medication seems likely. 
Definite (Causal):  
This relationship suggests that a definite causal relationship exists between 
the Treatment administration and the AE, and other conditions (concurrent 
illness, progression/expression of disease state, or concurrent medication 
reaction) do not appear to explain the event. 
 
Assessment of Severity 
The intensity of the AE will be rated as mild, moderate, or severe using the following criteria: 
Mild:  
These events require minimal or no treatment and do not interfere with the participant’s 
daily activities. 
Moderate: These events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 

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Severe:  
These events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually incapacitating. 
Changes in the severity of an AE should be documented to allow an assessment of the duration of 
the event at each level of intensity to be performed. Adverse events characterized as intermittent 
require documentation of onset and duration of each episode. 
 
Action taken to Adverse Event  
The action taken regarding the study treatment (corneal cross-linking) must be clarified in the CRF 
as follows: 
- 
None.  
- 
Study product not applied. 
- 
Unknown. 
 
Adverse Event treatment required 
The need for any treatment to solve the adverse event must be clarified in the CRF as follows: 
- 
None (other than protocol therapy). 
- 
Drug therapy. 
- 
Non-drug therapy. 
- 
Hospitalization. 
 
Outcome of Adverse Event 
The outcome of an event will be classified as follows:  
- 
Recovered.  
- 
Recovered with sequelae.  
- 
Ongoing.  
- 
Fatal.  
- 
Unknown/Lost to follow-up.  
 
Serious Adverse Event Reporting  
Any AE considered serious by the principal investigator of the Site of Investigation must be reported 
to IEC and the Sponsor within 24 hours from the time site personnel first learn about the event and 
during normal business CET hours.  
A written report must be submitted within 24 hours of the initial reporting to the IEC and the Sponsor 
and should consist of the Serious Adverse Event Report Form, accompanied by the following CRF 

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pages: the demographics page(s), the medical history page(s), the AE page(s) and the concomitant 
medications page(s). If the participant is hospitalized because of or during the course of an SAE, 
then a scanned copy of the hospital discharge summary should be sent by email to Sponsor as soon 
as it becomes available. Withdrawal from the study and all therapeutic measures will be at the 
discretion of the principal investigator. All SAEs (related or unknown relationship to the study 
Treatment) will be followed until satisfactory resolution or until the principal investigator or 
investigator deems the event to be chronic or the participant to be stable.  
The Principal Investigator of Site of Investigation will be responsible for telephone or fax reporting of 
any SAE to the IEC. Site will notify the IEC by telephone or fax transmission of any unexpected, 
fatal, or life-threatening experience (expedited report) associated with the use of the study Treatment 
as soon as possible but no later than 7 calendar days after the initial receipt of the information. Initial 
notification will be followed by a written report within 15 calendar days. For unexpected events 
associated with the use of the study Treatment, Sponsor/CRO will notify the IEC as soon as possible, 
but no later than 15 days of the initial receipt of information. The principal investigator is responsible 
for informing the Ethical Committee. Copies of SAE correspondence with the principal investigator 
or investigators, governing authorities, ethics committees, and the sponsor must be submitted to 
Sponsor/CRO for filing. 
 
Adverse Event Monitoring 
A participant experiencing any AE or SAE will receive treatment and follow-up evaluations by the 
principal investigator of the Site of Investigation or will be referred to another appropriate physician 
for treatment and follow-up. All AEs, whether serious or non-serious, should be followed to resolution 
or until the AE is determined by the investigator not to be clinically significant. 
 
List of adverse events reported in the specialized literature after CXL treatment 
Summary of adverse events on total 9607 cases treated by CXL procedures (ref. bibliography) 
N 
Type of adverse event 
Intensity of adverse event 
Cases (n.) 
Incidence (%) 
1 
Bacterial keratitis 
Severe 
59 
0.61% 
2 
Herpetic keratitis 
5 
0.05% 
3 
Corneal burn 
1 
0.01% 
4 
Corneal scarring 
51 
0.53% 
5 
CDVA loss (>2 ETDRS lines) 
13 
0.13% 

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6 
Persistent corneal haze 
Moderate 
12 
0.12% 
7 
Corneal infiltrates 
54 
0.56% 
8 
Epithelial ingrowth under LASIK flap 
1 
0.01% 
9 
Eyelid eczema 
Mild 
1 
0.01% 
10 
Transient eye pain 
18 
0.19% 
11 
Transient ocular itching 
1 
0.01% 
12 
Transient visual fogging 
16 
0.17% 
13 
Dry eye 
6 
0.06% 
14 
Conjunctival hyperemia 
10 
0.10% 
15 
Transient corneal haze 
499 
5.19% 
16 
Transient corneal edema 
74 
0.77% 
 
The table above shows the adverse events reported in studies of eyes treated both by Epi-Off and 
Epi-On corneal cross-linking procedures. In these studies, the majority of AEs generally developed 
in the short-term. The percentage of AEs that resolved by 3 months study visit was 98%. The most common 
AEs (i.e., ≥10%) were corneal epithelium defect, corneal opacity, corneal edema/striae, eye pain, 
and punctate keratitis. Most of these events represented sequelae following corneal epithelial 
debridement. Indeed, the Epi-Off corneal cross-linking procedure involves removal of the corneal 
epithelial layer, which is expected to result in reduction of vision due to the injury to the epithelium. 
The reduction in visual function and other adverse reactions generally resolve with the healing of the 
epithelium by the first week after treatment, though visual recovery to baseline values may take up 
to 3 months after treatment.  
 
Treatment strategy for adverse events 
Any adverse event is monitored in order to respond promptly and adequately to each of them. Since 
all participants will administer antibiotic/disinfectant eye drops and lubricant eye drops immediately 
after dosing and treatment, the majority of adverse event is managed preventively according to the 
standard of care after corneal cross-linking. The use of anti-inflammatory eye drops may be required 
in the case of moderate or severe ocular surface inflammation in a participant. The use of 
paracetamol pills may be used in the event of unacceptable headache in a participant. 
 
 

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Statistical Considerations and Data Analysis 
Stratification, Allocation and Enrolment 
Eligible participants will be stratified with allocation ratio 1:1 into either theranostic-guided CXL 
treatment protocol using a computer-generated stratification plan with blocks. Two different blocks 
are created, which include eyes with Kmax steeper or flatter than 54.0 D to stratify patients with 
comparable baseline Kmax values in either theranostic-guided CXL treatment protocol.  
This is a multi-center clinical trial. The stratification code is given to each local site of investigation 
by the central monitoring site of the Sponsor after the participant has been considered eligible to the 
study and has signed the informed consent. The enrolment is competitive. The period for enrollment 
includes the four months after study protocol approval by the IEC of the local site of investigation.  
 
Synopsis on disposition of participants 
 
 
 
Sample size 
By expecting a response rate of 50% of cases that will reach the threshold of -1.05 D of the Kmax 
value, a sample size of 42 patients achieves 91% power to detect a difference of 0,25 between the 
area under the ROC curve (AUC) under the null hypothesis of 0,60 and an AUC under the alternative 
hypothesis of 0,85 using a two-sided z-test at a significance level of 0,05000. The data are 
continuous responses. Considering a ≤20% drop-out rate (determined from systematic literature 
review on controlled and open clinical studies on standard and epithelium-on CXL procedures), a 
number of 50 participants (25 per treatment protocol) is allowed to be enrolled in the study. 
Details on the methodology and process used to define the sample size of this clinical trial are 
described in Appendix 1 – Methodology for sample size calculation.   
Eligible participant 
enrolled 
Investigational Epi-OFF 
corneal cross-linking
Investigational Epi-ON 
corneal cross-linking

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Statistical methodology 
Descriptive statistics will be used to summarize the data: numerical variables will be summarized as 
mean and standard deviation or median and interquartile range, respectively, according to the 
distribution of the data; categorical variables will be represented as frequencies and proportions. 
Student’s t-test or Wilcoxon test will be applied to compare continuous variables, while Fisher's exact 
test or Chi-Squared test will be used to analyze categorical variables. Bonferroni correction is applied 
to analysis of exploratory outcome measures of stratification groups. For all reported AEs, the data 
can be presented with events listed in order of decreasing frequency. 
The optimal cut-off values in predicting the propensity of CXL treatment to flatten the Kmax value at 
12-months was determined based upon the results of preclinical studies (data not disclosed to 
investigators to avoiding methodological study bias) and can be optimized with the Youden index.  
The accuracy and precision (95% CI) of the combined use of the theranostic imaging biomarkers to 
predict CXL treatment outcome will be determined by calculating the proportion of correctly classified 
eyes and the positive predictive value (PPV) respectively. The miss rate, which is the probability that 
a true positive is missed by the theranostic methodology, will be determined by calculating the false 
negative rate (FNR). 
Statistical significance will be set at 0.05. All the analysis will be performed using the statistical 
software R (latest version available). 
 
Population to be analysed 
The Intent-to-Treat (ITT) Population consists of all participants who were enrolled into the trial and 
performed at least 1 follow-up visit. 
The Per Protocol (PP) Population will be a subset of the ITT population and will consists of all 
participants who participated and completed at least 66% of all the visits (i.e., 4 of 6 total visits) and 
the last visit 6 at day-360 and in whom there are no major protocol deviations.  
Both populations will be agreed upon by Sponsor, Principal Investigator/Site of Investigation after 
the final subject visit and in advance of the database lock. 
 
 
 

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Ethical and Regulatory Issues 
This study adheres with the ethical principles that have their origin in the Declaration of Helsinki 
(Seventh revision, 2013), the Convention of Oviedo, April, 4th 1997, and is consistent with GCP, 
including the ICH Guideline for good clinical practice E6, the Directive 2001/20/EC, the ISO 
14155:2011, the Regulation (EU) 536/2014 of the European Parliament and of the Council of 16 
April 2014, the Regulation (EU) 2017/745 of the European Parliament and of the Council of 5 April 
2017, the Ministerial decree of the Italian Minister of Health of 2 August 2005, the Decree n.211 of 
24 June 2003, the Ministerial Decree of 14 July 2009 and applicable regulatory requirements 
concerning human subject protection and other aspects of clinical investigations.  
The study is conducted in strict compliance with the study protocol; any revision to study protocol 
must be discussed with and prepared by the Sponsor. The Investigator should not implement any 
deviation or change to the protocol without prior review and documented approval/favorable opinion 
from the Sponsor and IEC of an amendment. Any significant deviation must be documented in the 
CRF. 
 
Good Clinical Practice (GCP) for Clinical trials 
The Study will be conducted in accordance with the 13 principles of the rules of good clinical practice, 
as described below: 
1) The study complies with the principles of the Declaration of Helsinki. 
2) The expected benefits to participants justify the risks. 
3) The rights, safety, and well-being of the participants to the trial are the most important 
considerations and prevail over those of science and society. 
4) The available information about the investigational product are adequate to support the 
proposed clinical trial. 
5) The clinical study is drafted according to strong technical and scientific background and is fully 
described in the study protocol. 
6) The study will be conducted in accordance with the study protocol only after approval of the 
ethical committee. 
7) The medical care given to, and the medical decisions made on behalf of the participants will 
always fall under the responsibility of qualified medical specialists. 
8) All the investigators have the education, training, and experience to perform their specific tasks. 
9) An informed consent should be freely obtained by each participant prior to his participation in 
the study. 

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10) Any information concerning the clinical study is recorded, handled, and stored in a way that 
allows its accurate reporting, interpretation and verification. 
11) The confidentiality of the documents is ensured by respecting the privacy and confidentiality 
rules provided for by the applicable regulatory requirements. 
12) The investigational products are manufactured, handled, and stored in compliance with good 
manufacturing practice applicable and are used according to the approved protocol. 
13) Procedures are implemented to guarantee the quality of every aspect of the trial. 
 
Independent Ethics Committee approval 
The International Conference on Harmonisation (ICH) guidelines require that approval be obtained 
from an independent ethical committee (IEC) prior to participation of human participants in research 
studies.  Prior to the study onset, the protocol, informed consent, advertisements to be used for 
participant recruitment, and any other written information regarding this study to be provided to the 
participant must be approved by the IEC. Documentation of all IEC approvals and of the IEC 
compliance with ICH Guideline E6 will be maintained by the site and will be available for review by 
the sponsor or its designee. 
The Principal Investigator/Site of Investigation and CRO, if applicable, will support the Sponsor in 
answering question from the IEC in relation to the study or study procedures. 
All IEC approvals should be signed by the IEC Chairman or designee and must identify the IEC 
name and address, the clinical protocol by title and/or protocol number and the date approval and/or 
favorable opinion was granted. 
The study may only begin after approval of the same committee. Before initiating the clinical trial, 
the Principal Investigator/Site of Investigation should have written and dated approval/favorable 
opinion from the IEC for the trial protocol. 
 
Informed consent  
A written informed consent (Annex 3) shall be obtained from each participant prior to entering the 
study or performing any unusual or non-routine procedure that involves risk to the participant.  An 
informed consent template may be provided by the sponsor to investigative sites. If any institution-
specific modifications to study-related procedures are proposed or made by the site, the consent 
should be reviewed by the sponsor and/or its designee, if appropriate prior to IEC submission.  Once 
reviewed, the consent will be submitted by the Principal Investigator/Site of Investigation to the IEC 
for review and approval prior to the start of the study. If the informed consent form is revised during 
the course of the study, all active participating participants must sign the revised form. 

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Before recruitment and enrollment, each prospective participant will be given a full explanation of 
the study and allowed to read the approved informed consent form. Once the Principal 
Investigator/Site of Investigation is assured that the participant understands the implications of 
participating in the study, the participant will be asked to give consent to participate in the study by 
signing the informed consent form. 
The Principal Investigator/Site of Investigation shall provide an original form of the signed informed 
consent to the participant. A second original form shall be maintained in the participant’s medical 
records at the site. 
 
Risk Assessment and Management 
The present study does not accept healthy volunteers. Participants who are eligible to the study are 
those affected by keratoconus. 
This particular study design assumes that the theranostic-guided riboflavin/UV-A corneal cross-
linking treatment does not induce in the participants unacceptable risks. Several clinical 
studies10,11,12,13,14 have shown safety and efficacy of corneal cross-linking using a 0.2% riboflavin 
ophthalmic solution to protect the cornea prior to UV-A irradiation. The occurrence of severe adverse 
events, such as ulcerative keratitis, have been registered in 0.2% of Epi-OFF CXL procedures; no 
severe adverse events have been recorded after Epi-ON CXL procedures. 
The safety of participants has been assessed according to the following issues: 
1. 
The use of CE marked medical devices for the indication of use; 
2. 
the risks inherent in the type and route of administration of the products; 
The seriousness of possible adverse reactions and the probability of them happening have been 
considered during study design and listed under “Adverse Events” section. The innovative corneal 
cross-linking procedure is expected to elevate the gold standard practice by improving predictability 
of CXL clinical outcome and thus to provide adequate therapeutic benefit to justify entering 
keratoconus patients into the study. The management of the possible adverse reactions has been 
described under “Adverse Events” section. 
The Sponsor has given the Principal Investigator/Site of Investigation and IEC full access to the 
relevant pre-clinical work on the medical devices in the Investigator’s Brochure (Annex 8). The 
 
10 Zhang X, Sun L, Chen Y, Li M, Tian M, Zhou X. One-year outcomes of pachymetry and epithelium thicknesses after accelerated 
(45 mW/cm2) transepithelial corneal collagen cross-linking for keratoconus patients. Sci Rep 2016;6:32692. 
11 Artola A, Piñero DP, Ruiz-Fortes P, Soto-Negro R, Pérez-Cambrodí RJ. Clinical outcomes at one year following keratoconus treatment 
with accelerated transepithelial cross-linking. Int J Ophthalmol 2017;10(4):652-655.  
12 Tian M, Jian W, Sun L, Shen Y, Zhang X, Zhou X. One-year follow-up of accelerated transepithelial corneal collagen cross-linking for 
progressive pediatric keratoconus. BMC Ophthalmol 2018;18(1):75.  
13 Kır MB, Türkyılmaz K, Öner V. Transepithelial high-intensity cross-linking for the treatment of progressive keratoconus: 2-year outcomes. 
Curr Eye Res 2017;42(1):28-31.  
14 Huang J, Shen Y, Jian W, Xu H, Li M, Zhao J, Zhou X, Liao H. Two-year topographic and densitometric outcomes of accelerated 
(45 mW/cm2) transepithelial corneal cross-linking for keratoconus: a case-control study. BMC Ophthalmol 2018;18(1):337. 

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medical devices have been shown to be designed and manufactured according to the Directive 
93/42/CE and to be further safe for the indication of use in pre-clinical and regulatory testing. 
The Sponsor ensures for the entire duration of the study to monitor the safety of subjects by strict 
assessment of the risks inherent in the type of medical device and its target and in any potential 
immediate toxicity predicted from non-clinical or literature information. 
 
 
 
 

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Sites of Investigation 
• 
Azienda Ospedaliero Universitaria Careggi – Università di Firenze 
The Careggi University Hospital has a public legal personality and is empowered with 
entrepreneurial, organizational, administrative, patrimonial, financial, managerial and technical 
autonomy. It is a Trust integrated with the University of Florence and is characterized by the health 
care activities of hospitalization, specialized outpatient services and Emergency Room/Urgent care 
services. It pursues the development of high standard specialization and is a Reference in the 
Florentine Wider Area as well as on a regional and national scale. The Corneal Unit of the 
Ophthalmology department is the clinical unit involved in the present clinical study. The Unit is 
engaged in the validation of new methods of diagnosis and treatment of corneal and anterior 
segment eye diseases under the responsibility of Dr. Rita Mencucci. Both the research activities and 
the clinical and surgical activities are carried out at the at main Careggi University Hospital site. 
• 
Azienda Ospedaliera Universitaria Policlinico G. Martino – Università di Messina 
The G. Martino University hospital has a public legal personality and is a Trust integrated with the 
University of Messina. It is characterized by the health care activities of hospitalization, specialized 
outpatient services and Emergency Room/Urgent care services. It pursues the development of high 
standard specialization and is a Reference in the regional scale. The Corneal Unit of the 
Ophthalmology department is the clinical unit involved in the present clinical study. This Unit is 
engaged in the validation of new methods of diagnosis and treatment of ocular surface, corneal and 
anterior segment eye diseases under the responsibility of Dr. Anna Maria Roszkowska. Both the 
research activities and the clinical and surgical activities are carried out at the the Policlinico G. 
Martino. 
• Azienda Ospedaliera Universitaria Mater Domini – Università Magna Graecia di Catanzaro  
The Mater Domini University hospital has a public legal personality and is a Trust integrated with the 
University of Catanzaro. It is a modern structure pursuing the development of high standard 
specialization and is a Reference in the regional scale. The Ophthalmology department is the clinical 
unit involved in the present clinical study. The department is engaged in the validation of new 
methods of diagnosis and treatment of eye diseases under the responsibility of Prof. Vincenzo 
Scorcia.  
 

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Investigator qualifications 
• 
Prof. Rita Mencucci graduated in Medicine and Surgery at the University of Florence in 1984 
with 100/100 and honors. She specialized in Ophthalmology at the University of Florence in 
1988 with full marks and honors. She is Professor at the school of ophthalmology of the 
University of Florence. Member of numerous scientific societies. Author of numerous 
publications in peer-reviewed scientific journals, she has also been a speaker at numerous 
national and international conferences. Her main area of expertise includes keratoconus 
management and corneal cross-linking.  
• 
Prof. Anna Maria Roszkowska graduated in Medicine and Surgery in 1987 at the University 
Jagiellonia of Cracovia (Poland) with 110/110 cum laude. She holds a PhD in Refractive Surgery 
at the University of Padua in 1999. She is associate professor of Ophthalmology at the University 
of Messina. Author of numerous publications in peer-reviewed scientific journals, she has also 
been a speaker at numerous national and international conferences. Her main area of expertise 
includes keratoconus management and corneal cross-linking.  
• 
Prof. Vincenzo Scorcia graduated in Medicine and Surgery at the University of Rome “La 
Sapienza” in 2001 with 110/110 and honors. He specialized in Ophthalmology at the University 
of Magna Græcia of Catanzaro in 2006 with 90/90 and honors. He is author of numerous 
publications in peer-reviewed scientific journals and a speaker at numerous national and 
international conferences. He has been researcher at the Faculty of Medicine and Surgery of 
the University of Magna Græcia of Catanzaro since 2008. He has been Associate Professor, 
Director of the Ophthalmology Unit and the Specialization School in Ophthalmology since 2016. 
 
 
 
 
 
 

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Sponsor 
Regensight s.r.l. (hereinafter “Regensight” or the “startup company”) was established in 26 July 2019 
in order to develop and introduce the theranostics technology in eye care. 
The company’s legal form is a limited liability company. It is registered with n. RM-1586517 in the 
special section for innovative startup Co. in the Chamber of Commerce of Roma, VAT n. 
IT15376761001. 
The legal representative of Regensight is Dr. Marco Lombardo. Dr. Marco Lombardo was graduated 
with honours in Medicine and Surgery and specialized with honours in Ophthalmology. He holds a 
PhD in Biomedical Engineering and Computer Science. He has developed several innovative 
medical devices for the diagnosis and treatment of eye diseases, including adaptive optics retinal 
imaging, corneal iontophoresis for treating keratoconus, scleral iontophoresis for the prevention of 
AMD progression, femtosecond laser for cataract surgery. He has been working as senior researcher 
at an ophthalmic IRCCS in Rome for 9 years. He has been one of expert members of the Technical 
Scientific Direction of the Italian Ministry of Health, member of the expert committee for the clinical 
application of new technologies in Visual Science of OSA, member of the Association of Research 
in Vision and Ophthalmology (ARVO), the European Society of Cataract and Refractive Surgery 
(ESCRS) and the Società Italiana di Oftalmologia (SOI). He published more than 100 papers in peer-
reviewed scientific journals. He is inventor of 3 patent families.  
 
 

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Administrative Rules 
Clinical data handling 
The Sponsor is responsible for communicating the start, end and eventual discontinuation of the trial 
to the Regulatory Authorities and Independent Ethical Committees (IEC).  
The Clinical Investigators are required to ensure that the data collected during the study protocol are 
accurate, complete, verifiable and traceable. In addition, they are obliged to inform the IEC and the 
Sponsor of all serious and unexpected adverse events related to medical devices used in the study, 
in accordance with the Legislative Decree n. 211/2003.  
The Clinical Investigators are required to send to the Sponsor a detailed final report on the outcome 
thereof. The Sponsor and Principal Investigator are required to write a final report on the study 
outcome, which will be forwarded to the IECs. 
The Sponsor is required to notify the IECs about any changes to the present study protocol.  
Before commencing the present study, the Sponsor and the Sites of Investigation will sign a Clinical 
Trial Agreement (Annex 10). The identification codes and the CRF of each participant recruited in 
the present study will be archived by the Sponsor and the Site of Investigation until the time limit set 
by law. 
 
Case Report Forms 
As part of the responsibilities assumed by participating in the study, the clinical investigators agree 
to maintain adequate case histories for the participants treated as part of the research under this 
protocol. The clinical investigators agree to maintain accurate CRFs (Annex 1) and source 
documentation as part of the case histories according to the instructions to complete CRF (Annex 
2). The Sponsor will supply the CRFs. All CRFs should be completed legibly in black ink. The CRFs 
may not be completed in pencil. 
All CRF information is to be filled in.  If an item is not available or is not applicable, this fact should 
be indicated. Blank spaces should not be present unless otherwise directed.  A correction should be 
made by striking through the incorrect entry with a single line and the corrected information should 
be entered adjacent to the deleted item. The correction must be initialed and dated by the person 
making the correction. 
Each completed CRF must be reviewed, signed, and dated by the principal investigator and clinical 
monitor in a timely manner. The completed CRF will be checked by study monitors as soon as 
practical after completion. Scan copies of the CRF pages required to elaborate the interim report will 
be sent to the statistician responsible for the analysis of data during the study. At the end of the study 

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the site will send a scanned copy of all CRF to the Sponsor and will retain the originals at the site in 
the principal investigator’s files. 
 
Reports 
Each site of Investigation will provide information about study progression to the Sponsor on a three 
months basis after the enrolment of first patient into the study. This information will be reported by 
the Sponsor in the form (Annex 5) provided by the Sponsor to the Principal Investigator/Site of 
Investigation.  
In addition, each site of Investigation shall write, in English, an interim report (Annex 6) including all 
patients who have completed visit 5 (day 180), according to the ICH E3 guidance on structure and 
content of clinical study reports. 
Each form must be filled with the following information:   
• 
number of subjects visited at baseline (and/or reason for enrolment failure) (visit 1); 
• 
number of subjects recruited (visit 1); 
• 
number of subjects receiving the treatment (day 0); 
• 
Average intracorneal concentration of riboflavin after dosing (day 0). 
• 
Average intracorneal concentration of riboflavin at the end of treatment (day 0). 
• 
Average Treatment Efficacy Index (day 0). 
• 
Number of subjects completing visits after treatment. 
• 
number of premature terminations/drop-outs. 
• 
Average UDVA at each visit. 
• 
Average CDVA at each visit. 
• 
Average manifest refraction at each visit. 
• 
Average ECD at each visit. 
• 
Slit lamp biomicroscopy data at each visit. 
• 
Interim analysis of primary outcome measure at 6-months. 
• 
Adverse events (number and type). 
• 
Missing data. 
• 
Other information (if any). 
A final report (Annex 7) shall be elaborated in written (English) according the ICH E3 guidance on 
structure and content of clinical study reports and sent to the Sponsor for approval. The Final Report 
will compile all the data collected from all the participants during the entire duration of the study (from 
screening to the end of follow-up, including AE (if any), discussion of statistical analysis as well as 
interpretation of the results. This report will be sent to the Sponsor within 2 weeks from the final 

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monitoring visit performed by the Sponsor to confirm the correctness and completeness of the data 
collected and database lock. In the Final Report, a clear conclusion on the following points must be 
provided: 
• 
Assessment of primary outcome measure at 12 months (day 360), such as: 
- 
Validation of the combined use of the theranostic imaging biomarkers. True positives, 
false positives, true negatives and false negatives were evaluated in order to 
determine the ability of the theranostic imaging biomarkers, including both the 
riboflavin score and theranostic score, to predict the propensity of CXL treatment in 
flattening the Kmax value (D) by more than 0.1 D at 12 months postoperatively. 
• 
Assessment of all secondary outcome measures, such as: 
- 
Change of Maximum Keratometry assessed by corneal topography at 12-months. 
- 
Change of Endothelial Cell Density assessed by specular microscopy at 12-months. 
• 
Assessment of the additional exploratory outcome measures, such as: 
- 
Change of Corrected Distance Visual Acuity (CDVA) assessed by ETDRS at 12-
months. 
- 
Change of Uncorrected Distance Visual Acuity (UDVA) assessed by ETDRS at 12-
months. 
- 
Change of Manifest Refraction assessed by ETDRS at 12-months. 
- 
Change of secondary outcome measures at 12-months in either stratification group. 
 
Study Reporting Requirements 
By participating in this study, the Principal Investigator/Site of Investigation agree to provide the 
Sponsor with the adequate study reports, as defined in the “Reports” section above.  
The Principal Investigator/Site of Investigation agree to submit reports of AEs and SAEs according 
to the timeline and method outlined in the protocol. In addition, the Principal Investigator/Site of 
Investigation agree to submit a report to the IEC as appropriate.   
 
Monitoring of the Study  
The Site of Investigation has the obligation to follow the study closely and to support the by Sponsor 
in monitoring the study. In doing so, the Principal Investigator/Site of Investigation will assist the 
Sponsor to visit the study facility as defined in the monitoring plan, in addition to maintaining 
necessary contact through telephone, email, and letter. The Sponsor will maintain current personal 
knowledge of the study through observation, review of study records and source documentation, and 
discussion of the conduct of the study with the principal investigator or staff. All aspects of the study 

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must be carefully monitored for compliance with applicable government regulation with respect to 
current GCP and current standard operating procedures. 
 
Monitoring Plan 
On-site monitoring will be performed by the Sponsor at the Site of Investigation before commencing 
the study and at the end of the study (after visit 6, i.e., 360 days after all participants have been 
treated). On-site monitoring will be carried out to verify that: 
• 
the Principal Investigator receives the current Investigator's Brochure, all documents, and all 
trial supplies needed to conduct the trial properly and to comply with the applicable regulatory 
requirement(s).  
• 
the Principal Investigator and Study Staff have adequate experience and have been 
adequately informed and trained to carry the study. 
• 
the Site of Investigation has adequate resources to carry the study. 
• 
the investigators follows the approved protocol and all approved amendment(s). 
• 
written informed consent was obtained before each subject's participation in the trial.  
• 
the storage of the RitSight™ are acceptable, and that supplies are sufficient throughout the 
trial.  
• 
the disposition of unused investigational product(s) at the trial sites complies with applicable 
regulatory requirement(s) and is in accordance with the sponsor.  
• 
the RitSight™ is supplied only to subjects who are eligible to receive it and at the protocol 
specified doses.  
• 
the CRF entries and other operating documents are filled with accuracy and completeness by 
Investigators.  
• 
If CRFs have any entry error, omission, or illegibility, informing the investigator of it. The 
Sponsor shall ensure that appropriate corrections, additions, or deletions are made, dated, 
explained (if necessary), and initialed by the principal investigator.  
• 
all adverse events are appropriately reported within the time periods required by GCP, the 
protocol, the IEC, the Sponsor, and the applicable regulatory requirement(s).  
• 
if deviations from the protocol occur, communicating the applicable regulatory requirements to 
the investigator and taking appropriate action designed to prevent recurrence of the detected 
deviations.  
Centralized monitoring will be performed by the Sponsor to review interim and final reports provided 
by the Principal Investigation/Site of Investigation. Review will include analyses of study data, 
including the following actions:  

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• 
to identify missing data, inconsistent data, data outliers, unexpected lack of variability and 
protocol deviations.  
• 
to evaluate for systematic or significant errors in data collection and reporting at a site or across 
sites; or potential data manipulation or data integrity problems.  
• 
to perform statistical analysis of study data. 
 
Inspection of Records 
The Principal Investigator/Site of Investigation will permit, at any working day, trial-related 
monitoring, audits, IEC review, and regulatory inspection(s) by providing direct access to all study 
records. In the event of an audit, the Principal Investigator/Site of Investigation agrees to allow the 
Sponsor, representatives of the sponsor, the Regulatory authorities to access to all study records. 
The Principal Investigator/Site of Investigation should promptly notify the Sponsor of any audits 
scheduled by any regulatory authorities and promptly forward copies of any audit reports received 
to the sponsor. 
 
Study Record Retention 
Essential documents should be retained until at least 5 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing applications 
in an ICH region or at least 3 years have elapsed since the formal discontinuation of clinical 
development of the investigational product.  These documents should be retained for a longer period, 
however, if required by the applicable regulatory requirements or by an agreement with the Sponsor.  
It is the responsibility of the Sponsor to inform the Principal Investigator/Site of Investigation as to 
when these documents no longer need to be retained. 
 
Quality Assurance 
The Sponsor is responsible for implementing and maintaining quality assurance and quality control 
systems to ensure that the study is conducted and data are generated, documented and reported in 
compliance with the protocol, GCP and the applicable regulatory requirements. The Sponsor 
ensures that case report forms, and other operational documents are clear, concise, and consistent. 
The Sponsor shall make sure that each C4V CHROMO4VIS™ system and each batch of the 
RitSight™ eye drop meets the requirements and that is properly delivered to the Site of Investigation. 
The Principal Investigator/Site of Investigation ensures the accuracy, completeness, legibility, and 
timeliness of the data reported to the Sponsor in the CRFs and in all required reports. The Principal 
Investigator/Site of Investigation ensures that there is adequate training for all staff participating in 

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the conduct of the study, and that they are aware of regulatory requirements and acceptable 
standards for the conduct of clinical trials and the protection of human subjects. 
The Sponsor ensures to monitor that all operating documents are using standard terminology and to 
provide systematic safeguard to ensure adherence to study protocol by the Principal Investigator/Site 
of Investigation. 
 
Confidentiality 
All evaluation forms, reports, and other records will be identified in a manner designed to maintain 
participant confidentiality. All records will be kept in a secure storage area with limited access.  
Clinical information will not be released without the written permission of the participant [or the 
participant’s legal guardian], except as necessary for monitoring and auditing by the sponsor, its 
designee, the regulatory authorities, or the IEC. 
The Principal Investigator/Site of Investigation shall not disclose or use for any purpose other than 
performance of the study, any data, record, or other unpublished, confidential information disclosed 
to those individuals for the purpose of the study. Prior written agreement from the Sponsor must be 
obtained for the disclosure of any said confidential information to other parties. 
 
Personal data protection 
The study adheres to the General Data Protection Regulation (GDPR). The rights of each participant 
to the study to physical and mental integrity, to privacy and to the protection of the data (“personal 
data”) concerning him/her in accordance with data protection laws, are safeguarded. Personal data 
include any information relating to an identified or identifiable natural person ('data subject'); an 
identifiable person is one who can be identified, directly or indirectly, in particular by reference to an 
identification number or to one or more factors specific to his physical, physiological, mental, 
economic, cultural or social identity. The Sponsor monitors whether the Principal Investigator/Site of 
Investigation performs the study in conformity with the protocol and safeguard personal data. In this 
study, data records are key-coded data and thus any other person cannot de-code the data easily. 
The Sponsor and the Principal Investigator/Site of Investigation will take appropriate actions to 
safeguard personal data of the study. The data controller is the legal representative of the Sponsor; 
the data processor is the Principal Investigator. 
 
Modification of the Protocol 
Any changes in this research activity, except those necessary to remove an apparent, immediate 
hazard to the participant, must be reviewed and approved by the Sponsor. Amendments to the 

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protocol must be submitted in writing to the IEC for approval prior to participants being enrolled into 
an amended protocol. 
 
Protocol Violations and Deviations 
The Principal Investigator/Site of Investigation must document and explain in the participant’s source 
documentation any deviation from the approved protocol. The Principal Investigator/Site of 
Investigation may implement a deviation from, or a change of, the protocol to eliminate an immediate 
hazard to trial participants without prior IEC approval. As soon as possible after such an occurrence, 
the implemented deviation or change, the reasons for it, and any proposed protocol amendment(s) 
should be submitted to the IEC for review and approval, to the sponsor for agreement, and to the 
regulatory authorities, if required. 
A deviation from the protocol is an unintended and/or unanticipated departure from the procedures 
and/or processes approved by the Sponsor and the IEC and agreed to by the Principal 
Investigator/Site of Investigation. Deviations usually have an impact on individual participants or a 
small group of participants and do not involve inclusion/exclusion or primary endpoint criteria.  A 
protocol violation occurs when there is nonadherence to the protocol that results in a significant, 
additional risk to the participant, when the participant or Principal Investigator/Site of Investigation 
has failed to adhere to significant protocol requirements (inclusion/exclusion criteria) and the 
participant was enrolled without prior sponsor approval, or when there is nonadherence to authorities 
regulations. 
The Sponsor will document protocol violations and deviations throughout the course of monitoring 
visits.  The Sponsor will notify the principal investigator during a visit and/or in writing of all violations 
and deviations. The IEC should be notified of all protocol violations and deviations in a timely manner. 
 
List of major deviations/violation 
• 
Patient not met inclusion criteria 
• 
Patient not met exclusion criteria 
• 
Not obtained signed consent form from patient 
• 
Corneal topography not done at visit 1 (baseline) 
• 
Corneal topography not done at visit 6 (day 360) 
• 
Corneal cross-linking treatment not completed 
 
 
 

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Financial Disclosure and Obligations 
The clinical investigators are required to provide financial disclosure information to allow the Sponsor 
to submit the complete and accurate certification or disclosure statements to regulatory authorities. 
In addition, the principal investigator or investigators must provide to the Sponsor a commitment to 
promptly update this information if any relevant changes occur during the course of the investigation. 
The clinical investigators are required to provide an updated curriculum vitae (CV).  
The Sponsor is not financially responsible for further testing/treatment of any medical condition, 
which may be detected during the screening process. In addition, in the absence of specific 
arrangements, the Sponsor is not financially responsible for further treatment of the participant’s 
disease. 
 
Study Conduct 
The Principal Investigator/Site of Investigation agree that the study will be conducted according to 
the principles of the ICH E6 Guideline for Good Clinical Practice and the principles of the World 
Medical Association Declaration of Helsinki. The Principal Investigator/Site of Investigation will 
conduct all aspects of this study in accordance with all national, state, and local laws or regulations. 
 
Recruitment strategies 
The screening and the recruitment processes will respect the dignity and autonomy of the potential 
participants by avoiding any potential undue influence and by protecting both the privacy of the 
individual and the confidentiality of any information obtained for recruitment and/or screening 
purposes.  
In preparing recruitment materials the Principal Investigator/Site of Investigation shall consider the 
purpose of the research and the setting in which the research will be conducted. The following 
methods of recruiting subjects will be considered acceptable: 
- 
Direct recruitment of potential study participants 
Examples of this strategy are physicians talking with their own or clinic patients about the study, 
contact between the study team and potential subjects in person, on the phone or on the internet. 
With this method considerable care will have to be taken so that the person contacted does not feel 
pressured to participate.  
- 
Referrals 
Referrals may be from non-investigator healthcare providers, participants referring other 
participants.  

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- 
Advertisements, flyers, information sheets, notices, internet postings and/or media can be used 
to recruit subjects.  
Prospective participants who respond to these will contact directly the Site of Investigation. 
Recruitment materials can contain the following Information: 
1. The name of the institution(s); 
2. The name of the Principal Investigator(s) and Sponsor, and the name of a contact person 
with a telephone number and email address to call and write for information about the study; 
3. The purpose of the research and, in summary form, the eligibility criteria that will be used to 
admit subjects into the study; 
4. The location of the research and time commitment, if appropriate. 
The Promoter and Sponsor can prepare advertisements, information sheets, notices, internet 
postings and/or media but cannot directly contact prospective subjects (see Annex 12). Per-
participant incentive payments or referral fees, whether paid for each referral or each enrollment, are 
not allowed. 
 
Publications 
Clinical data are the property of the Sponsor and cannot be published without prior authorization 
from the Sponsor, but data and publication thereof will not be unduly withheld. 
The data of the study may be considered for reporting at a scientific meeting or for publication(s) in 
a scientific journal. In these cases, the Sponsor will be responsible for these activities and will decide 
who will eventually present study outcomes at a scientific meeting, if and how the manuscript(s) is 
written and edited, the number and order of authors, the scientific journal(s) to which it/they will be 
submitted, and other related issues. The Sponsor has final approval authority over all such issues.   
 
Study Termination 
The Sponsor may prematurely terminate the study in case of major violations, which may prevent 
the safety of the subjects or future evaluation of the study results. In addition, the Sponsor, may 
prematurely terminate the study, in its sole discretion and for any reason whatsoever associated with 
changes in its commercial or clinical strategy.  
In such an occurrence the Sponsor shall give at least thirty (30) calendar days’ written notice of 
termination to the study centre. All the effort will be made to perform a final visit to all the subjects 
still in the study aiming to confirm their wellbeing. 
 

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Alio JL, Abbouda A, Valle DD, Del Castillo JM, Fernandez JA. Corneal cross-linking and 
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Angunawela RI, Arnalich-Montiel F, Allan BD. Peripheral sterile corneal infiltrates and melting 
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Baenninger PB, Bachmann LM, Wienecke L, Kaufmann C, Thiel MA. Effects and adverse 
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Chan E, Snibson GR. Current status of corneal collagen cross-linking for keratoconus: a 
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Chan E, Snibson GR. Current status of corneal collagen cross-linking for keratoconus: a 
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9. 
Chunyu T, Xiujun P, Zhengjun F, Xia Z, Feihu Z. Corneal collagen cross-linking in keratoconus: 
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Dhawan S, Rao K, Natrajan S. Complications of corneal collagen cross-linking. J Ophthalmol 
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Doughty MJ, Cullen AP. Long-term effects of a single dose of ultraviolet-B on albino rabbit 
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Fantes FE, Hanna KD, Waring III GO, Pouliquen Y, Thompson KP, Savoldelli M. Wound 
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14. 
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Godefrooij DA, Roohé SL, Soeters N, Wisse RPL. The Independent Effect of Various Cross-
Linking Treatment Modalities on Treatment Effectiveness in Keratoconus. Cornea 2020;39:63–
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Gordon-Shaag A, Millodot M, Shneor E, Liu Y.  The Genetic and Environmental Factors for 
Keratoconus. Biomed Res Int 2015; 2015: 795738. 
17. 
Greenstein SA, Fry KL, Bhatt J, Hersh PS. Natural history of corneal haze after collagen 
crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J 
Cataract Refract Surg 2010; 36: 2105-2114. 
18. 
Kinsey VE. Spectral transmission of the eye to ultraviolet radiations. Arch Ophthalmol 1948; 
39:508-513. 
19. 
Koller T, Mrochen M, Seiler T. Complication and failure rates after corneal crosslinking. J 
Cataract Refract Surg 2009; 35: 1358-1362. 
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Appendix 1 - Methodology for sample size calculation  
Sample size calculation has been based on a thorough literature search on clinical investigations of 
the state of the art on corneal cross-linking for the treatment of keratoconus.  
The inclusion criteria included: 
A. Population: patients affected by keratoconus  
B. Intervention: corneal cross-linking 
Outcome/endpoint: change of maximum keratometry (Kmax) measured with corneal topography. 
The exclusion criteria included: 
C. lack to report adequate information on study methodology (population, intervention, statistics); 
D. follow-up shorter than 1-year follow-up after corneal cross-linking. This approach guarantees to 
analyze pertinent data with enough follow-up time to assess safety and performance of the UV-
A medical device for the indication of use. 
E. present case report or small (<15 participants) case series or have been published as a letter to 
the Editor; 
F. are retrospective clinical studies; 
G. have not been published or have been published in scientific journals without impact factor or in 
journal with impact factor lower than 1.5 (i.e., the last quartile of the JcR category 
“Ophthalmology” in 2018) This approach guarantees that proper scientific methodology has 
been used in order to drive significant conclusions on the results; 
H. have been published earlier than 2013. 
The search output provided the following scientific articles: 
1. Chunyu T, Xiujun P, Zhengjun F, Xia Z, Feihu Z. Corneal collagen cross-linking in keratoconus: 
a systematic review and meta-analysis. Sci Rep 2014; 4: 5652.  
2. Craig JA, Mahon J, Yellowlees A, Barata T, Glanville J, Arber M, Mandava L, Powell J, Figueiredo 
F. Epithelium-off photochemical corneal collagen cross-linkage using riboflavin and ultraviolet a 
for keratoconus and keratectasia: a systematic review and meta-analysis. Ocul Surf 
2014;12(3):202-214. 
3. Hersh PS, Stulting RD, Muller D, Durrie DS, Rajpal RK, United States Crosslinking Study Group. 
United States multicenter clinical trial of corneal collagen crosslinking for keratoconus treatment. 
Ophthalmology 2017; 124(9): 1259-1270.  
4. Hersh PS, Stulting RD, Muller D, Durrie DS, Rajpal RK, U.S. Crosslinking Study Group. U.S. 
Multicenter clinical trial of corneal collagen crosslinking for treatment of corneal ectasia after 
refractive surgery. Ophthalmology 2017; 124(10): 1475-1484.  

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5. Jiang LZ, Jiang W, Qiu SY. Conventional vs. pulsed-light accelerated corneal collagen cross-
linking for the treatment of progressive keratoconus: 12-month results from a prospective study. 
Exp Ther Med. 2017 Nov;14(5):4238-4244. doi: 10.3892/etm.2017.5031. Epub 2017 Aug  
6. Kır MB, Türkyılmaz K, Öner V. Transepithelial High-Intensity Cross-Linking for the Treatment of 
Progressive Keratoconus: 2-year Outcomes. Curr Eye Res 2017;42(1):28-31. 
7. Kobashi H, Rong SS. Corneal Collagen Cross-Linking for Keratoconus: Systematic Review. 
Biomed Res Int. 2017;2017:8145651 
8. Kobashi H, Tsubota K. Accelerated versus standard corneal cross-linking for progressive 
keratoconus: a meta-analysis of randomized controlled trials. Cornea 2020; 39(2): 172-180.  
9. Li J, Ji P, Lin X. Efficacy of corneal collagen cross-linking for treatment of keratoconus: a meta-
analysis of randomized controlled trials. PLoS One. 2015 May 18;10(5):e0127079. 
10. Mazzotta C, Traversi C, Paradiso AL, Latronico ME, Rechichi M. Pulsed Light Accelerated 
Crosslinking  versus Continuous Light Accelerated Crosslinking: One-Year Results. J Ophthalmol 
2014; 2014: 604731.  
11. McAnena L, Doyle F, O'Keefe M. Cross-linking in children with keratoconus: a systematic review 
and meta-analysis. Acta Ophthalmol 2017;95(3):229-239.  
12. Meiri Z, Keren S, Rosenblatt A, Sarig T, Shenhav L, Varssano D. Efficacy of Corneal Collagen 
Cross-Linking for the Treatment of Keratoconus: A Systematic Review and Meta-Analysis. 
Cornea 2016;35(3):417-428.  
13. Shajari M, Kolb CM, Agha B, Steinwender G, Müller M, Herrmann E, Schmack I, Mayer WJ, 
Kohnen T. Comparison of standard and accelerated corneal cross-linking for the treatment of 
keratoconus: a meta-analysis. Acta Ophthalmol 2019;97(1):e22-e35.  
14. Tomita M, Mita M, Huseynova T. Accelerated versus conventional corneal collagen crosslinking. 
J Cataract Refract Surg 2014;40(6):1013-1020.  
15. Wen D et al. Comparison of epithelium-Off versus transepithelial corneal collagen cross-Linking 
for keratoconus: a systematic review and meta-analysis. Cornea 2018;37(8):1018-1024.  
16. Wen D, Li Q, Song B, Tu R, Wang Q, O'Brart DPS, McAlinden C, Huang J. Comparison of 
Standard Versus Accelerated Corneal Collagen Cross-Linking for Keratoconus: A Meta-
Analysis. Invest Ophthalmol Vis Sci 2018;59(10):3920-3931. 
17. Zhang X, Zhao J, Li M, Tian M, Shen Y, Zhou X. Conventional and transepithelial corneal cross-
linking for patients with keratoconus. PLoS One 2018; 13(4): e0195105.  
 
 
 

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The analysis of pertinent data from 6.995 cases treated by corneal cross-linking has shown an 
average Kmax change of -1.05±0.80 D after 1 year follow-up, showing that the treatment is 
effective for stabilizing the cornea, which has been weakened by disease or laser surgery. 
The average Kmax change was -0.79±0.75 D at 6-month follow-up. 
 
 
A further search was done to understand whether the CXL treatment protocol (i.e., standard CXL or 
transepithelial CXL) may influence the change of Kmax at 1-year follow-up.  
The inclusion criteria included: 
I. 
Population: patients affected by keratoconus  
J. 
Intervention: corneal cross-linking 
K. Type of study: randomized controlled trial 
Outcome/endpoint: change of maximum keratometry (Kmax) measured with corneal topography. 
The exclusion criteria included: 
L. 
lack to report adequate information on study methodology (population, intervention, statistics); 
M. have follow-up shorter than 1-year follow-up after corneal cross-linking. This approach 
guarantees to analyze pertinent data with enough follow-up time to assess safety and 
performance of the UV-A medical device for the indication of use. 
N. present case report or small (<15 participants) case series or have been published as a letter to 
the Editor; 
O. are retrospective clinical studies; 
P. are meta-analysis studies; 
Q. have not been published or have been published in scientific journals without impact factor or in 
journal with impact factor lower than 1.5 (i.e., the last quartile of the JcR category 
“Ophthalmology” in 2018) This approach guarantees that proper scientific methodology has 
been used in order to drive significant conclusions on the results. 
R. Have been published earlier than 2013. 
The search output provided the following scientific articles: 
1. 
Hersh PS et al. United States Crosslinking Study Group. United States multicenter clinical trial 
of corneal collagen crosslinking for keratoconus treatment. Ophthalmology 2017; 124(9): 1259-
1270. 
2. 
Wittig-Silva et al. A Randomized, controlled trial of corneal collagen cross-linking in progressive 
keratoconus. Three-Year Results. Ophthalmology 2014; 121:812-821.  

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3. 
Soeters N et al. Transepithelial versus epithelium-off corneal cross-linking for the treatment of 
progressive keratoconus: a randomized controlled trial. Am J Ophthalmol 2015;159(5):821-8.e3.  
4. 
Hashemian H et al. Evaluation of corneal changes after conventional versus accelerated corneal 
cross-linking: a randomized controlled trial. J Refract Surg 2014;30(12):837-42.  
5. 
Lombardo M et al. Randomized controlled trial comparing transepithelial corneal cross-linking 
using iontophoresis with the Dresden protocol in progressive keratoconus. Ophthalmology 2017; 
124: 804-812. 
6. 
Al Fayez MF et al. Transepithelial versus epithelium-off corneal collagen cross-linking for 
progressive keratoconus: a prospective randomized controlled trial. Cornea 2015; 34 Suppl 
10:S53-6.  
 
 
The analysis of pertinent data from the above articles provided the following results: 
• 
354 cases have been treated by standard corneal cross-linking showing an average Kmax 
change of -1.41±1.71 D after 1 year follow-up; the average Kmax change was -0.89±0.60 
D at 6-month follow-up. 
• 
91 cases have been treated by transepithelial corneal cross-linking showing an average 
Kmax change of was -0.32±1.55 D after 1 year follow-up; the average Kmax change was -
0.52±1.20 D at 6-month follow-up. 
• 
In 122 control, no treated cases, the natural history of keratoconus progression has shown 
a mean change of Kmax +0.89±2.70 D during 1-year follow-up; the average Kmax change 
was +0.93±1.35 D at 6-month follow-up.