Multimodal Retinal Imaging in the Detection and Follow-up of Alzheimer's Disease
Conditions: Alzheimer Disease, Alzheimer Dementia, Mild Cognitive Impairment, Dementia, Glaucoma, Age-Related Macular Degeneration, Diabetic Retinopathy
Sponsor: Universitaire Ziekenhuizen KU Leuven

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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Protocol Full Title prospective observational trial: 
S60932 Multimodal retinal imaging in the detection and follow-up of Alzheimer’s disease 
 
Protocol Acronym/short title: 
RetAD 
 
Version and date of protocol: 
 v 1.2 27/10/2017 
 
 
Sponsor: 
 
Name: UZLeuven 
Address: Herestraat 49, 3000 Leuven 
 
Principal Investigator: 
 
Name: Prof. Dr. Ingeborg Stalmans 
Address: Kapucijnenvoer 33, campus Sint-Rafaël, dienst oftalmologie  
Telephone: +32 16 33 23 72 
Email: ingeborg.stalmans@uzleuven.be 
 
Co-Investigator: 
 
Name: Prof. Dr. Rik Vandenberghe 
Address: Herestraat 49 – bus 7003 48, campus Gasthuisberg, dienst neurologie 
Telephone: +32 16 34 42 80 
Email: rik.vandenberghe@uzleuven.be 
 
 
 
 
S60932 Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal 
retinal imaging in the detection and follow-up of Alzheimer’s 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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Table of Contents 
Table of Contents .................................................................................................................................... 3 
1. 
Study Synopsis ................................................................................................................................. 5 
2. 
Background, rationale and novelty ................................................................................................. 7 
3. 
Trial objectives and design .............................................................................................................. 8 
3.1 
Preparation, organization and management of phase 1 study ........................................... 9 
3.2 
Preparation, organization and management of phase 2 study ......................................... 12 
3.3  Preparation, organization and management of longitudinal follow-up ................................ 13 
4. 
Selection and withdrawal of subjects ........................................................................................... 15 
4.1 Inclusion criteria ...................................................................................................................... 15 
4.2 Exclusion criteria ..................................................................................................................... 15 
4.3 Expected duration of trial ........................................................................................................ 16 
5. 
Trial procedures............................................................................................................................. 16 
5.1 
MRI and 18F-flutemetamol PET-CT scans ........................................................................... 16 
5.2 
Sample collection and handling ........................................................................................ 16 
5.3 
Genetic analysis ................................................................................................................. 16 
5.4 
Fasting laboratory test analysis ......................................................................................... 16 
5.5 
Ocular exams ..................................................................................................................... 16 
5.6 
Summary of visits/procedures .......................................................................................... 17 
5.7 
Supervision and responsibilities ........................................................................................ 17 
5.7.1 
Insurance ................................................................................................................... 18 
5.7.2 
Informed consent ...................................................................................................... 18 
6. 
Assessment of efficacy .................................................................................................................. 18 
7. 
Assessment of safety ..................................................................................................................... 18 
8. 
Statistics ........................................................................................................................................ 19 
8.1 Sample size .............................................................................................................................. 19 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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8.2 Analysis .................................................................................................................................... 19 
9. 
Quality assurance .......................................................................................................................... 19 
10. 
Direct access to source data and documents ............................................................................ 19 
11. 
Ethics and regulatory approvals ................................................................................................ 19 
12. 
Data Handling ............................................................................................................................ 20 
13. 
Data Management ..................................................................................................................... 20 
14. 
Translational research ............................................................................................................... 20 
15. 
Publication Policy ...................................................................................................................... 21 
16. 
Financial Aspects ....................................................................................................................... 21 
17. 
References ................................................................................................................................. 22 
 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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1. Study Synopsis  
 
Title of clinical trial 
 
 
Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
Protocol Short Title/Acronym 
 
 
RetAD 
 
Sponsor name 
 
 
UZLeuven 
 
Principal Investigator 
 
 
Prof. Dr. Ingeborg Stalmans 
Medical condition or disease under 
investigation 
 
Alzheimer’s disease 
 
Purpose of clinical trial 
 
 
To evaluate the use of non-invasive, multimodal retinal 
imaging for the early detection of Alzheimer’s disease 
and for the evaluation of disease progression 
 
Primary objective 
 
 
To identify retinal biomarkers for Alzheimer’s disease 
by means of non-invasive, multimodal retinal imaging 
Secondary objective(s) 
 
 
- 
To evaluate the diagnostic performance of 
selected ocular biomarkers for Alzheimer’s 
disease 
- 
To deliver a proof-of-concept for the use of 
retinal biomarkers to quantitatively measure 
cerebral Aβ load 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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- 
To deliver a proof-of-concept for the use of 
retinal biomarkers to follow Alzheimer’s 
disease progression 
 
Trial Design 
 
 
Observational, cross-sectional and prospective cohort 
 
Endpoints 
 
 
- 
Technical capability to detect Alzheimer’s 
disease by means of non-invasive retinal 
imaging 
- 
Technical capability to follow up Alzheimer’s 
disease progression by means of non-invasive 
retinal imaging 
- 
If acceptable technical capability 
demonstrated: determination of diagnostic 
accuracy of multimodal retinal imaging in 
Alzheimer’s disease (sensitivity and specificity) 
 
Sample Size 
 
 
90 individuals  
(35 amyloid-negative, 55 amyloid-positive) 
Summary of eligibility criteria 
 
 
• 
Age between 50 and 85
• 
In stable medical condition and willing and able
 
 to perform study procedures.  
• 
Fluent in written and verbal Dutch. 
 
• 
Capable of giving informed consent. 
 
Maximum duration of treatment of a 
subject 
 
/ 
 
Version and date of final protocol 
 
 
v 1.2  27/10/2017 
 
Version and date of protocol amendments 
 
 
 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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2. Background, rationale and novelty 
 
Alzheimer’s disease (AD) is the most common neurodegenerative disorder and the leading cause of 
dementia worldwide.1 A growing number of people are surviving into their 80s-90s and the number of 
AD patients projected to nearly triple in the next three decades, affecting 80-90 million people 
worldwide by 2040.1,2 As such, AD will become the third cause of death for older people, just behind 
cardiovascular disease and cancer. In contrast to the latter, AD cannot be prevented, slowed or cured. 
AD represents an enormous socio-economic burden and has become a trillion dollar disease.3 Despite 
decades of intensive research, diagnosis and treatment remain challenging for AD. A string of recent 
failures in clinical trials for AD drugs has pointed out that our understanding of the disease is still far 
from complete. More in detail, three major reasons underlying this treatment gap have been 
identified:  
i. The lack of techniques for patient screening and early diagnosis.  
ii. The incomplete understanding of the complex interplay of pathological processes that underlie 
AD.  
iii. The many hurdles between drug discovery and approval.   
 
With this study, we propose a novel way to address these needs, by using the retina as a model organ 
to study the central nervous system (CNS). Many of the hallmark cerebral pathophysiological processes 
of AD have also been observed in the retina. Unlike the rest of the CNS, the retina can be visualized 
directly, with an imaging resolution up to 100x higher than PET and MR scans. Using these high-
resolution imaging tools such as Optical Coherence Tomography (OCT), studies have demonstrated 
microvascular changes and neuro-retinal thinning in AD patients. Pilot data show that retinal Aβ can 
be visualized non-invasively solely based on the intrinsic hyperspectral signature of aggregated 
amyloid deposits.4 Non-invasive retinal imaging (e.g., fundus photography, OCT, hyperspectral imaging 
(HSI)) – which are all available at affordable cost –, could therefore represent novel means for 
identifying patients at risk, for longitudinal follow-up of disease progression in AD patients, and for 
research in a quest for more effective treatments. 
 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
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3. Trial objectives and design 
 
The ambition of this study is to contribute to AD diagnosis and clinical research via noninvasive 
multimodal imaging of the retinal manifestation of AD. It aims to respond to four problems that are 
currently being faced within the AD research field, by delivering a proof-of-concept for the use of 
multimodal imaging of retinal biomarkers for diagnosis and disease staging of AD, thereby tackling:  
1. the lack of an affordable, multimodal retinal imaging platform for clinical research and 
practice;  
2. the difficulties in recruiting Aβ-positive presymptomatic patients for phase III clinical trials;  
3. the need for non-invasive markers to monitor disease progression from a presymptomatic 
stage of the disease onwards;  
4. the lack of integrative prognostic software for disease identification.  
The main contribution will consist of the examination of the validity of retinal biomarkers as a 
diagnostic measure for Aβ-positivity, and the determination of their ability to quantitatively measure 
change in Aβ load. At the end of the project, we aim to deliver a research setup of a hyperspectral 
camera for retinal imaging, standardized image acquisition protocols for retinal imaging of AD 
biomarkers using different technologies (including hyperspectral imaging) and a data science approach 
for AD diagnosis. Proof-of-concept for the predictive value of multimodal imaging of selected retinal 
biomarkers in diagnosing and classifying AD patients will be delivered.  
This may be in the future useful for screening, early diagnosis and follow-up of AD. This is of major 
importance because AD has a long preclinical and prodromal phase, and by the time symptoms 
suggestive of a clinical diagnosis appear, neurodegeneration may have led to damage too extensive to 
repair.   
The primary objective of the human clinical diagnostic trial is to validate the retinal biomarkers against 
Aβ load measured with 18F-flutemetamol PET as standard-of-truth. The cross-sectional data collection 
is mainly aimed at exploring retinal biomarkers as a diagnostic measure for Aβ-positivity, while 
gathering the longitudinal information is mainly aimed at determining their ability to quantitatively 
measure change in Aβ load. The discriminative value of retinal imaging will be assessed (phase 1) and 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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subsequently evaluated (phase 2). All patients will be followed longitudinally over the course of 24 
months. 
This is an open-label longitudinal biomarker study without investigational medicinal product in 
subjects in different stages of the AD spectrum. 
The data that we will collect consist of amyloid imaging, MRI, blood, genetic, general health and 
cognitive data, as well as visual acuity, ocular biomicroscopy and funduscopy, fundus photographs, 
hyperspectral retinal images, Optical Coherence Tomography (OCT) retinal images and OCT 
angiography (OCT-A) retinal images. Subjects will be followed longitudinally. In the current study we 
will primarily investigate the potential of non-invasive, multimodal retinal imaging for the early 
detection of Alzheimer’s disease and for the evaluation of disease progression. This will be done in 
comparison with amyloid imaging and neuropsychological evaluations. 
We will build a longitudinal database of ocular, systemic, neuro-psychiatric, MRI and PET imaging 
parameters of Aβ-positive and Aβ-negative patients with different stages of cognitive impairment. This 
database will be used to provide proof-of-concept that retinal biomarkers provide an early, accurate 
and non-invasive tool for AD detection and follow-up. All data will be collected in a database for 
statistical analysis.   
3.1 Preparation, organization and management of phase 1 study 
 
The primary objective of this exploratory phase 1 study is to evaluate which ophthalmological 
measures optimally differentiate between Aβ-positive, clinically probable AD patients and Aβ-
negative, cognitive-intact healthy controls, matched for age, education, gender and apoE status.  
Methodology  
Study cohorts:  
1. 
Fifteen clinically probable AD patients, according to the National Institute of Aging (NIA)-
Alzheimer's Association (AA) diagnostic criteria,5 with an MMSE of 12 or higher, and with a positive 
CSF biomarker analysis or a positive amyloid-PET scan in the past or a positive amyloid-PET scan 
at inclusion, will be included in this group. Patients will be recruited via the memory clinic UZ 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
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Leuven, which is headed by Prof. dr. Rik Vandenberghe (total number of dementia patients due 
to AD currently in follow-up = 486).  
2. 
Fifteen Aβ-negative cognitive-intact healthy controls will be recruited from a longitudinal cohort 
of 180 cognitively healthy older adults who are followed at the Laboratory for Cognitive Neurology 
of Prof. dr. Rik Vandenberghe (UZ Leuven) (mean age = 68.4 ± 6.4, range 55-80 years).6,7 These 
community-dwelling participants were first recruited between 2010 and 2015 for multimodal 
brain imaging (Aβ PET and volumetric MRI) combined with neuropsychological assessment. One 
of the key inclusion criteria was neuropsychological test scores within the published norms at 
baseline. This cohort has been well characterized at baseline by means of detailed evaluation of 
lifestyle factors (lifelong cognitive and physical activity), quantitative parameters (weight, height, 
blood pressure under standardized conditions), cognitive reserve score (Cognitive Reserve 
Questionnaire), and neuropsychological performance. Participants are longitudinally followed 
with neuropsychological assessments at two-yearly intervals. The ready availability of this deeply 
phenotyped cohort is a strength of this project.   
Aβ-negative cognitive-intact controls will be matched to the AD patients for age, gender, 
education and apoE status. The Aβ-negative status and the cognitive-intact status will be verified 
by a repeat Aβ PET scan and a neuropsychological assessment at inclusion in the current study.  
Study procedures:  
After signing the informed consent, patients will be asked to fill in a questionnaire. All eligible 
participants will then undergo detailed ophthalmological examination, an MRI scan, 18F-flutemetamol 
PET scan, blood sampling (apoE genotyping and fasting laboratory tests – Table 1), as well as a detailed 
neuropsychological assessment.   
Hematology
Chemistry
Genotyping
Hematocrit
Gamma GT
apoE polymorphism
Hemoglobin
Alkaline phosphatase
Platelet count
Alanine aminotransferase (ALT)
WBC (total and differential)
Aspartate 
aminotransferase 
(AST) 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
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Sedimentation rate
Bicarbonate
Calcium
Chloride
Potassium
Sodium
Creatinin
Urea
Low-density Lipoprotein
C-reactive protein
Glycemia
HbA1c
Table 1: Laboratory tests 
- Ophthalmological examination will include visual acuity measurement, biomicroscopy and 
funduscopy, followed by the acquisition of hyperspectral, (angio-) OCT, and fundus photography using 
predetermined protocols.  
- PET images will be acquired on a TruePoint Siemens PET scanner using static acquisition during an 
acquisition window of 30 minutes, starting 90 minutes post-injection. A low-dose computed 
tomography scan will be performed for attenuation correction, prior to the PET scan. The summed 18F-
flutemetamol PET image (sumPET) will be co-registered to the subjects’ T1-weighted MRI image. 
Segmentation parameters from the subjects T1-weighted MRI will be used to normalize the co-
registered sumPET. A Standardized Uptake Value Ratio (SUVR) image will be created by dividing the 
normalized sumPET image by the uptake in the cerebellar grey matter reference region. SUVR values 
will then be calculated in a composite volume of interest (VOI) (SUVRcomp), consisting of frontal, 
parietal, anterior cingulate, posterior cingulate and lateral temporal VOIs, masked by the subject-
specific gray matter map.6  
- MRI will be performed on a 3T Achieva dstream Philips MRI scanner with a 32-channel head coil. The 
MRI sequence will consist of an MPRAGE volumetric MRI, and a FLAIR and Gradient Echo sequence.  

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
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- The Neuropsychological test battery will consist of the Auditory Verbal Learning Test (AVLT), 
Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Raven’s Progressive 
Matrices (RPM), Mini Mental State Examination (MMSE), Neuropsychiatric Inventory (NPI), and Cornell 
Scale for Depression in Dementia (CSDD). 
Statistical analysis:  
The primary statistical analysis will be based on a Receiver Operating Characteristic analysis of the 
different ophthalmological measures, to determine which have the best diagnostic accuracy (area 
under the curve) compared to the binary Aβ PET classification as standard-of-truth. In the cross-
sectional studies, the standard-of-truth will consist of the binary distinction between Aβ-positive cases 
and Aβ-negative cases, based on Aβ PET. This distinction will rely on a semi-quantitative analysis and 
a cut-off based on independent data.7 The phase 1 study is considered exploratory and the diagnostic 
accuracy of a wide range of ophthalmological parameters will be assessed. The three ophthalmological 
parameters with the best diagnostic accuracy will be selected as primary outcome measures for further 
validation in the phase 2 study in an independent cohort.  
3.2 Preparation, organization and management of phase 2 study 
 
The primary objective of this study is to evaluate the diagnostic accuracy of three ophthalmological 
measures, selected based on the phase 1 study, for discriminating between Aβ-negative and Aβ-
positive healthy cognitive-intact older adults and for discriminating between Aβ-negative cognitive-
intact older adults and Aβ-positive MCI patients.  
Methodology  
Study cohorts:  
1. 
Patients with amnestic MCI due to AD (Aβ-positive MCI), according to the NIA-AA diagnostic 
criteria, an MMSE 24 or higher, and with a positive CSF biomarker analysis or a positive amyloid-
PET scan in the past or a positive amyloid-PET scan at inclusion, recruited via the memory clinic 
UZ Leuven (total number of amnestic MCI patients currently in follow-up = 64).   
2. 
Aβ-positive cognitive-intact older adults. These will be recruited from the longitudinal cohort of 
older adults followed at the Laboratory for Cognitive Neurology described above. At inclusion 

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(between 2010-2015) 30 out of 180 participants were Aβ-positive.  A positive CSF biomarker 
analysis or a positive amyloid-PET scan in the past or a positive amyloid-PET scan at inclusion is a 
prerequisite for inclusion in this group. 
3. 
Aβ-negative cognitive-intact older adults, recruited from the same longitudinal cohort.  
 
Study procedures:  
cfr. Phase 1 study  
Statistical analysis:  
The primary analysis will be based on a contingency table, in which cognitively intact subjects will be 
classified according to one ophthalmological parameter and compared to the classification based on 
the Aβ PET. This will be done for the three parameters that have been selected based on the phase 1 
data. The same will be done for the classification of the cognitively intact Aβ-negative cases versus the 
Aβ-positive MCI cases.  
3.3  Preparation, organization and management of longitudinal follow-up 
 
All participants of the phase 1 and phase 2 study (n = 90) will be invited to participate in the longitudinal 
component.   
Methodology  
Study procedures:  
Subjects will undergo 6-monthly ophthalmological evaluations. After 24 months, all baseline 
examinations will be repeated. For longitudinal studies, the optimal reference region has been shown 
to be subcortical white matter rather than cerebellum or pons.8 The calculated SUVRcomp values at 
follow-up (t24) will be subtracted from the SUVRcomp values at baseline (t0).  
Statistical analysis:  
The primary analyses will consist of a regression analysis between the change in the three selected 
ophthalmological parameters and the change in Aβ load. The second primary objective will be the 
correlation between the change in these three parameters and the change in the total learning and 

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the delayed recall of the AVLT. Secondary objectives will consist of a regression analysis between the 
change in the ophthalmological parameters and the change in the RBANS score, a regression analysis 
between the change in these parameters and the volumetric changes on the MRI, and a regression 
analysis between the change in the ophthalmological parameters and the changes in Aβ load analyzed 
in a voxelwise manner. In a further secondary analysis, the participants will be divided into Aβ 
accumulators and non-accumulators, based on the Aβ PET, and the ophthalmological parameters will 
be compared between these two groups.  
This project is financed by external funding (FWO SBO project, decision will be communicated in 
December 2017). 
 
 
 
 
 
 
 
Figure 1: study timeline 
Devices that will be used: 
- MRI: 3T Achieva dstream Philips MRI scanner with a 32-channel head coil, Philips, Netherlands 
- PET-CT: TruePoint Siemens PET scanner, Siemens, Germany 
- OCT: Spectralis, Heidelberg Engineering, Germany 
- Fundus picture: Topcon DRC 50-DX fundus camera, Topcon Corporation, Japan 
- Hyperspectral retinal imaging:  
XIMEA SNm4x4 hyperspectral snapshot camera, Ximea, Germany 
SNAPSCAN VNIR hyperspectral snapscan camera, Imec, Belgium 
 

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4. Selection and withdrawal of subjects 
 
4.1 Inclusion criteria 
 
Between ≥ 50 and ≤ 85 years of age. 
 
In the opinion of the investigator, the patient is in stable medical condition and willing and able to 
perform study procedures. 
 
Patient is fluent in written and verbal Dutch. 
 
Patient is capable of giving informed consent. 
4.2 Exclusion criteria 
 
Patient has a history or current evidence of a neurological disorder, which, in the opinion of the primary 
investigator, may contribute to the subject’s cognitive impairment. 
Patient has a history of large-vessel stroke or evidence of a large-vessel infarction or other focal lesions 
on baseline MRI scan, which may contribute to the cause of the memory impairment in the opinion of 
the investigator. Vascular white matter lesions or other signs of microangiopathy will not be 
considered an exclusion.  
Patient has a history of malignancy ≤ 5 years prior to signing informed consent, except for patients 
who have undergone potentially curative therapy with no evidence of recurrence for 1 year, and who 
are deemed at low risk for recurrency by her/his treating physician. 
Patient is currently participating or has participated in a study with an investigational compound within 
30 days of signing informed consent. 
Subject has any magnetizable metal prostheses, implants or foreign objects that could pose a hazard 
during MRI scans. 
Patient has a known history of ocular diseases other than the exception of cataract and/or wearing 
glasses/contact lenses. 

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4.3 Expected duration of trial 
 
First-patient first-visit is planned for January 2018, last-patient first-visit is planned for December 2018. 
Final data expected to be collected December 2020. 
5. Trial procedures 
 
5.1 MRI and 18F-flutemetamol PET-CT scans 
 
Cfr. Paragraph 3. 
5.2 Sample collection and handling 
 
Venous blood samples will be collected at start, and after 2 years in glass EDTA-containing (4), serum-
separating (2) and RNA Paxgene blood collection (1) tubes, and frozen according to specific 
instructions. Blood and serum samples will be stored in double at KU Leuven. 
5.3 Genetic analysis 
 
Samples for genotyping of the apoE polymorphism will be analyzed according to standard procedures 
by the Center for Human Genetics, UZ Leuven. 
5.4 Fasting laboratory test analysis 
 
Samples will be analyzed by the Department of Laboratory Medicine of UZ Leuven. 
5.5 Ocular exams 
 
Cfr. Paragraph 3. 
 
 
 

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5.6 Summary of visits/procedures 
 
Procedure
Timing 
Venous blood sampling
Hematology: Hematocrit, Hemoglobin, Platelet 
count, WBC (total and differential), 
Sedimentation rate 
t0, t24
Chemistry: Gamma GT, Alkaline phosphatase, 
Alanine aminotransferase (ALT), Aspartate 
aminotransferase (AST), Bicarbonate, Calcium, 
Chloride, Potassium, Sodium, Creatinin, Urea, 
Low-density Lipoprotein, C-reactive protein, 
Glycemia, HbA1c  
t0, t24
Genotyping: apoE polymorphism
t0
Brain MRI
MPRAGE volumetric MRI, en FLAIR and Gradient 
Echo sequence 
t0, t24
18F-Flutemetamol PET CT
t0, t24
Neuropsychiatric testing
Auditory Verbal Learning Test (AVLT)
Repeatable Battery for the Assessment of 
Neuropsychological Status (RBANS) 
Raven’s Progressive Matrices (RPM) 
Mini Mental State Examination (MMSE) 
Neuropsychiatric Inventory (NPI) 
Cornell Scale for Depression in Dementia (CSDD) 
t0, t24
Ocular examination
Visual acuity, biomicroscopy, funduscopy
Fundus pictures, including hyperspectral imaging 
OCT + angio-OCT 
t0, t6, 
t12, t18, 
t24 
Table 2: Summary of visits/procedures 
5.7 Supervision and responsibilities 
 
The study will be performed under the supervision of Profs. Ingeborg Stalmans and Rik Vandenberghe, 
UZ Leuven. The local study nurses will coordinate the logistics and take the blood samples. Ocular exams 
and retinal imaging will be performed by MD PhD students skilled with the procedures. The participating 
investigators will perform their part of the study fully in accordance with the terms of the Protocol, the 
applicable national laws (amongst others: the Belgian Law of May 7, 2004 relating to experiments on 
human persons; the Belgian Law of August 22, 2002 on Patient Rights; the Belgian Law of  December 8, 
Fasting 
laboratory tests 

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1992 (as amended) regarding the Protection of Privacy in relation to the Processing of Personal Data.) 
and apply and adhere to regulations and rules as, amongst others, the Declaration of Helsinki (2008) 
and ICH GCP Guidelines. 
 
5.7.1 
Insurance 
 
In accordance with the Belgian Law relating to experiments on human persons dated May 7, 2004, 
UZ/KULeuven shall assume, even without fault, the responsibility of any damages incurred by a Study 
Patient  from the UZ/KU Leuven site and linked directly or indirectly to the participation to the Study, 
and shall provide compensation therefore through their insurance. 
5.7.2 
Informed consent 
The Participating Site acknowledges that the Study can and will be conducted only on the basis of prior 
informed consent by the Subjects, or their legal representatives, to participate in the Study. The 
Participating Site shall obtain a signed informed consent form (ICF) for all patients prior to their 
enrollment and participation in the Study in compliance with all applicable laws, regulations and the 
approval of the (local) Ethics Committee, if required. The Participating Site shall retain such ICFs in 
accordance with the requirements of all applicable regulatory agencies and laws. 
6. Assessment of efficacy 
/ 
7. Assessment of safety 
 
The radiation exposure for the volunteers is for the 18F-flutemetamol scan (150 MBq target dose) 
maximally 5 mSv. 
No permanent adverse effects have been described after any of the planned ocular examinations. Only 
light from the visible spectrum is used. A temporary decreased vision due to the flashlight (generally 
less than 1 minute) is expected after the fundus picture. 

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8. Statistics 
8.1 Sample size 
 
Phase 1: based on previous trials on OCT measurements in AD and age-matched cognitive-intact 
controls,9 a sample size of 8 patients would be sufficient to discriminate both groups using a two-tailed 
t-test with alpha 0.05, a power of 0.90 and an estimated effect size of 2.73 µm (RFNL change). For this 
proof-of-concept study, we aim to recruit 15 patients per group.  
Phase 2: based a previous trial,10 comparing OCT measurements in Aβ-positive versus Aβ-negative 
cognitive-intact subjects, the target sample size is at least 13 patients per group (alpha = 0.05, power 
= 0.90, estimated effect size of 1.33). We aim to include 20 patients in each group.  
Longitudinal: all participants of the phase 1 and phase 2 study (n = 90) will be invited to participate in 
the longitudinal component.   
8.2 Analysis 
 
Cfr. Paragraph 3. 
9. Quality assurance 
 
To assure maximal quality and reproducibility, the trial protocol will be followed rigorously. 
10. 
Direct access to source data and documents 
 
Only data gathered in the context of the trial will be used.  
11. 
Ethics and regulatory approvals 
The trial will be conducted in compliance with the principles of the Declaration of Helsinki (2008), 
the principles of Good Clinical Practice and in accordance with all applicable regulatory 

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requirements. This protocol and related documents will be submitted for review to Ethics 
Committee of the University Hospitals Leuven. 
The Study will be conducted only on the basis of prior informed consent by the Subjects to 
participate in the Study. We shall obtain a signed informed consent form (ICF) for all patients prior 
to their enrollment and participation in the Study in compliance with all applicable laws, 
regulations and the approval of the (local) Ethics Committee, if required. We shall retain such ICFs 
in accordance with the requirements of all applicable regulatory agencies and laws. 
12. 
Data Handling 
 
We shall treat all information and data as confidential and shall not disclose such information to any 
third parties or use such information for any purpose other than the performance of the Study. The 
collection, processing and disclosure of personal data, will comply with applicable personal data 
protection and the processing of personal data (Directive 95/46/EC and Belgian law of December 8, 
1992 on the Protection of the Privacy in relation to the Processing of Personal Data). We will protect 
the data from disclosure outside the research according to the terms of the research protocol and the 
informed consent document. The subject’s name or other identifiers will be stored separately from 
their research data and replaced with a unique code to create a new identity for the subject. 
13. 
Data Management 
 
All data will be analyzed and stored in a coded fashion, with a unique anonymous identifier for every 
subject. In the future, these data could be used in the context of this project, and for related projects 
regarding cognitive functions, Alzheimer’s disease and related conditions.   
14. 
Translational research 
 
From every included patient, a blood sample will be collected and stored for genotyping and 
biochemical analysis.    

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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15. 
Publication Policy 
 
Any publication will be submitted to all co-authors at least thirty (30) days prior to submission or 
disclosure.  
16. 
Financial Aspects 
 
This project is financed by external funding (FWO SBO project, decision will be communicated in 
December 2017). 
All medical expenses (medication and  visits) arising from participation in the study will not be charged 
to the volunteer. Volunteers do not receive a financial compensation for their participation. 
Transportation costs and parking fees in the context of this study will be reimbursed accordingly, as 
well as the cost of a meal, with a maximum of 50 Euros per visit.   
 
 

Study protocol RetAD v 1.2 dd. 27/10/2017: Multimodal retinal imaging in the detection and 
follow-up of Alzheimer’s disease 
 
 
 
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17. 
References 
 
1. 
Weiner MW, Veitch DP, Aisen PS, et al. The Alzheimer's Disease Neuroimaging Initiative: A 
review of papers published since its inception. Alzheimer's & Dementia: The Journal of the 
Alzheimer's Association.9(5):e111-e194. 
2. 
2017 Alzheimer’s Disease Facts and Figures. Alzheimer’s Association;2017. 
3. 
Prince M, Wimo A, Guerchet M, Ali G-C, Wu Y-T, Prina M. World Alzheimer Report 2015 - The 
Global Impact of Dementia: An analysis of prevalence, incidence, cost and trends. London: 
Alzheimer's Disease International;2015. 
4. 
More SS, Beach JM, Vince R. Early Detection of Amyloidopathy in Alzheimer's Mice by 
Hyperspectral EndoscopyEarly Detection of Amyloidopathy in Alzheimer's Mice. Investigative 
Ophthalmology & Visual Science. 2016;57(7):3231-3238. 
5. 
McKhann GM, Knopman DS, Chertkow H, et al. The diagnosis of dementia due to Alzheimer’s 
disease: Recommendations from the National Institute on Aging-Alzheimer’s Association 
workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimer's & Dementia: The 
Journal of the Alzheimer's Association.7(3):263-269. 
6. 
Adamczuk K, Schaeverbeke J, Nelissen N, et al. Amyloid imaging in cognitively normal older 
adults: Comparison between 18F-flutemetamol and 11C-Pittsburgh Compound B. European 
journal of nuclear medicine and molecular imaging. 2016;43(1):142-151. 
7. 
Adamczuk K, De Weer A-S, Nelissen N, et al. Functional changes in the language network in 
response to increased amyloid β deposition in cognitively intact older adults. Cerebral Cortex. 
2014;26(1):358-373. 
8. 
Landau SM, Fero A, Baker SL, et al. Measurement of longitudinal β-amyloid change with 18F-
florbetapir PET and standardized uptake value ratios. Journal of Nuclear Medicine. 
2015;56(4):567-574. 
9. 
Ascaso FJ, Cruz N, Modrego PJ, et al. Retinal alterations in mild cognitive impairment and 
Alzheimer’s disease: an optical coherence tomography study. Journal of Neurology. 
2014;261(8):1522-1530. 
10. 
Snyder PJ, Johnson LN, Lim YY, et al. Nonvascular retinal imaging markers of preclinical 
Alzheimer's disease. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring. 
2016;4:169-178.