Non-invasive Transcutaneous Spectroscopy for the Assessment of Gut Permeability
Conditions: Development and Validation of Gut Permeability Sensor, Permeability; Increased, Celiac Disease, Inflammatory Bowel Diseases, Liver Diseases, HIV/AIDS
Sponsor: Imperial College London

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Non-invasive transcutaneous spectroscopy for the 
assessment of gut permeability 
 
 
Abbreviated title: GutPerm 
 
Protocol version 4.2A: 01-May-2019 
 
 
REC Reference: 18/LO/0714 
IRAS Project ID: 242462 
 
 
 
MAIN SPONSOR:   
 
 
Imperial College London  
 
 
FUNDER:  
 
 
 
Imperial College London 
 
 
STUDY COORDINATION CENTRE:  
Surgical Innovation Centre (Paterson Building), 
St. Mary’s Hospital, 
Imperial College London & Imperial College Healthcare NHS 
Trust, 
South Wharf Road, 
 
London, 
W2 1NY 
 
REC reference: 
 
 
18/LO/0714 
 
 
 
 
 
Protocol authorised by: 
 
Name & Role: 
Date: 
Signature: 
Dr Alex Thompson 
 
 
 

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CONTENTS 
 
CONTENTS 
2 
STUDY MANAGEMENT GROUP 
4 
SPONSOR  
5 
FUNDER 
 
6 
GLOSSARY OF ABBREVIATIONS 
6 
KEYWORDS 
6 
STUDY SUMMARY 
7 
Title 
7 
Design 
7 
Research questions 
7 
Outcome measures 
7 
Investigational medical devices 
7 
Contrast agents 
7 
Population 
8 
Eligibility 
8 
BACKGROUND 
9 
AIMS & OBJECTIVES 
11 
MATERIALS & METHODS 
11 
Instrumentation 
11 
Contrast agents 
13 
Patient/subject selection 
17 
Experimental protocols 
18 
Schedule of procedures 
26 
Safety Monitoring 
28 
Patient recruitment 
28 
INCLUSION CRITERIA 
29 
EXCLUSION CRITERIA 
29 
WITHDRAWAL OF SUBJECTS 
30 
ADVERSE EVENTS 
30 
Definitions 
30 
Reporting procedures 
30 
Non-serious AEs 
30 
Serious AEs 
30 
Contact details for reporting SAEs 
31 
SAMPLE SIZE & STATISTICAL ANALYSIS 
31 
DATA COLLECTION, DATA HANDLING & RECORD KEEPING 
33 
SAMPLE STORAGE 
34 
STOPPING/DISCONTINUATION RULES 
34 
RESEARCH GOVERNANCE, MONITORING, ETHICS AND R&D APPROVAL 
34 
Indemnity 
34 
Sponsor 
34 
Funding 
35 

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PUBLICATION POLICY 
35 
APPENDIX A 
36 
Optical setup and laser safety considerations for bench-top fluorescence spectrometer (‘mark I’) 
36 
APPENDIX B 
37 
Discussion of design and safety considerations for the miniaturised (‘mark II’) fluorescence spectrometer 
37 
REFERENCES 
38 
 
 
 

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STUDY MANAGEMENT GROUP 
 
 
Chief Investigator 
& Principal Investigator: 
Dr Alex Thompson 
Imperial College Research Fellow 
Surgical Innovation Centre (Paterson Building) 
Department of Medicine 
Imperial College London 
St Mary’s Hospital 
London 
W2 1NY 
alex.thompson08@imperial.ac.uk 
 
Co-Investigators: 
 
Professor Robert Goldin 
 
 
 
 
Professor of Gastro-intestinal and Liver Pathology  
 
 
 
 
Department of Cellular Pathology 
Imperial College London 
Clarence Wing 
St Mary's Hospital 
London 
W2 1NY 
r.goldin@imperial.ac.uk 
 
Professor Mark Thursz 
Professor of Hepatology 
Department of Surgery and Cancer 
Imperial College London 
Imperial College Healthcare NHS Trust 
St Mary’s Hospital 
London 
W2 1NY 
m.thursz@imperial.ac.uk 
 
Dr James Maurice 
 
 
 
 
Clinical Research Fellow 
 
 
 
 
Department of Surgery and Cancer 
Imperial College London 
Imperial College Healthcare NHS Trust 
St Mary’s Hospital 
London 
W2 1NY 
james.maurice@imperial.ac.uk 
 
Dr Jonathan Hoare 
Consultant Gastroenterologist 
Department of Surgery and Cancer 
Imperial College London 
Imperial College Healthcare NHS Trust 
St Mary’s Hospital 

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London 
W2 1NY 
jonathan.hoare@nhs.net 
 
Professor Francesca Cordeiro 
Professor of Ophthalmology 
Imperial College Ophthalmic Research Group (ICORG) 
Imperial College London 
Western Eye Hospital 
London 
NW1 5QH 
francesca.cordeiro@nhs.net 
 
Dr Aaron Lett 
Research and Teaching Fellow 
Nutrition and Dietetics Research Group  
Commonwealth Building 
Department of Medicine 
Imperial College London 
Hammersmith Hospital 
London 
W12 0NN 
a.lett@imperial.ac.uk 
 
 
Study Management:  
Dr Alex Thompson 
Imperial College Research Fellow 
Surgical Innovation Centre (Paterson Building) 
Department of Medicine 
Imperial College London 
St Mary’s Hospital 
London 
W2 1NY 
alex.thompson08@imperial.ac.uk 
 
 
SPONSOR 
 
Imperial College London is the main research sponsor for this study. For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at: 
 
 
 
Joint Research Compliance Office 
Imperial College London 
Room 221 
Medical School Building 
St. Mary’s Campus 
Norfolk Place 
London 
W2 1PG 
Tel: 020 7594 1872 

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FUNDER 
 
This study is funded by the Imperial College Research Fellowship scheme. 
 
 
GLOSSARY OF ABBREVIATIONS 
 
AE 
 
Adverse event 
EED 
 
Environmental enteric dysfunction 
FITC  
Fluorescein isothiocyanate 
GI 
 
Gastro-intestinal 
GER                Gastric emptying rate 
HIV 
 
Human immunodeficiency virus 
IBD 
 
Inflammatory bowel disease 
ICG 
 
Indocyanine green 
IMP 
 
Investigational medicinal product 
L:M 
 
Lactulose:Mannitol 
LED 
 
Light emitting diode 
MHRA  
Medicines and Healthcare products Regulatory Agency 
NRES  
National Research Ethics Service 
PD 
 
Photodiode 
PEG 
 
Polyethylene glycol 
REC 
 
Research Ethics Committee 
SAE 
 
Serious adverse event 
 
 
KEYWORDS 
 
Fluorescence, spectroscopy, gut permeability, non-invasive, low-cost 

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STUDY SUMMARY 
 
Title 
Non-invasive transcutaneous spectroscopy for the assessment of gut permeability 
 
Design 
Basic science study involving procedures with human participants 
 
 
 
 
 
Research questions 
Is it possible to use transcutaneous optical spectroscopy to non-invasively assess the 
permeability of the intestine via measurements of the concentration of orally administered 
contrast agents in the blood? 
 
Outcome measures 
Spectroscopic measurements of gut permeability (and gastric emptying rate, GER) will be 
assessed via comparison against a range of traditional markers: 
 
1. Where the blood concentration is known – for example, in ophthalmology patients who 
have received a direct intravenous dose of contrast agent – a direct comparison will be 
made without the need for further measurements. 
2. In subjects receiving an oral dose of contrast agent, blood/urine samples will be taken 
alongside the spectroscopic measurements in order to permit accurate ex vivo 
quantification of the serum/urine concentration in the laboratory. 
3. In control experiments aimed at determining the impact of GER, the rate of uptake of 
fluorescent dye will be compared against the rate of uptake of paracetamol (which is 
used clinically to assess GER). 
4. In patients who are also undergoing polyethylene glycol (PEG)-based permeability 
assays, spectroscopic permeability measurements will be compared to the results of 
the more traditional approach. 
5. Finally, in patients for whom intestinal biopsy and histology data is available, 
spectroscopic permeability measurements will be compared against histological 
measures of epithelial damage and permeability. 
 
Investigational medical devices 
The devices under investigation in this study are optical spectrometers designed for the 
transcutaneous measurement of the concentration of a fluorescent contrast agent in the blood. 
Two devices will be tested – a bench-top system that provides high level spectral information 
(‘mark I’) and a miniaturised wearable system suitable for wider clinical application (‘mark II’). 
The ‘mark II’ system will be designed during the study, based on results collected with the 
‘mark I’ device. As such, at this stage this research represents an investigation of a technique 
for assessment of gut permeability (transcutaneous fluorescence spectroscopy) rather than an 
investigation of a particular medical device. 
 
Contrast agents 
A variety of contrast agents will be tested in this study in order to find the most suitable agent(s) 
for transcutaneous quantification of gut permeability. The agents tested will include fluorescein, 
indocyanine green (ICG), fluorescein isothiocyanate (FITC) conjugated dextran (FITC-
dextran), and FITC-conjugated polyethylene glycol (FITC-PEG). For the FITC-dextran and 
FITC-PEG studies, contrast agents with varying molecular weights will be investigated. For 
GER experiments, the only contrast agent used will be fluorescein. 

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Population 
Three groups will be studied: 
 
Group 1 
Ophthalmology patients who are receiving an intravenous dose of either 
fluorescein or ICG as part of their routine ophthalmic care (e.g. as part of a 
fluorescence angiography examination). These patients will take part in 
preliminary studies aimed at determining whether it is possible to detect 
fluorescein and ICG in the blood using transcutaneous fluorescence 
measurements. 
 
Group 2 
Healthy subjects will be split into two groups (2a and 2b) dependent on the 
aspect of the study they participate in. 
                        
2a: Healthy subjects with no known issues of increased gut permeability. These 
subjects will act as negative controls in all gut permeability studies. A subset of 
these volunteers will be asked to take two spectroscopic permeability tests – 
one with and one without a supplementary dose of a hyperosmotic solution, 
which acts to transiently increase intestinal permeability. This subset will 
provide proof-of-concept data prior to recruitment of patients in Group 3. 
 
                       2b: Healthy subjects with no known issues of increased gut permeability or gut 
motility. These subjects will participate in GER experiments, which will provide 
complementary data to aid in the design of transcutaneous optical spectroscopy 
as a non-invasive tool to assess gut permeability. Separate “healthy volunteer” 
sub-group will prevent the possibility of build-up of contrast agent. 
 
Group 3 
Gastro-intestinal (GI) and non-GI patients who are expected to exhibit 
increased gut permeability (e.g. patients with coeliac disease, inflammatory 
bowel disease (IBD), liver disease, HIV or another condition in which increased 
intestinal permeability is common). The more extreme cases in this group will 
act as positive controls. 
 
Eligibility 
Aged 18 and over; able to give informed consent; not pregnant or breastfeeding; no prior 
adverse reactions to fluorescein, ICG, dextran, PEG or iodine. 
 
 
 

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BACKGROUND 
 
Increased gut permeability – often referred to as “leaky gut” – occurs when the intestinal barrier 
becomes compromised and involves increased transmission of certain intestinal constituents 
(such as endotoxins) into lymph and blood vessels [1, 2]. The condition is associated with 
many widespread diseases including coeliac disease [1, 3], inflammatory bowel disease (IBD) 
[3-5], human immunodeficiency virus (HIV) [6], fatty liver disease [7-9] and environmental 
enteric dysfunction (EED) [10, 11]. These diseases affect millions of patients worldwide and 
represent a huge cost burden to national healthcare systems. Importantly, in many of them, 
the role and impact of intestinal barrier function is not yet completely understood. Moreover, 
the methods used to assess permeability are cumbersome. As a result, increased permeability 
of the intestine has not yet been conclusively linked to outcomes in these illnesses [1]. 
 
The issue of increased intestinal permeability is particularly acute in the case of EED, a chronic 
inflammatory condition of the small intestine that impairs both nutrient absorption and barrier 
function [10, 11]. EED is endemic in many developing countries – where it manifests in children 
at a very early age (< 6 months) – and can lead to malnutrition, poor response to vaccines, 
and severely restricted physical and cognitive development [12-17]. As such, it is strongly 
associated with poor developmental and socio-economic outcomes including growth stunting, 
poor performance in education, reduced income in adulthood, and increased fertility [18-20]. 
 
Increased gut permeability is believed to play a pivotal role in EED [21, 22], where the leakage 
of pathogens (including endotoxins and, in some extreme cases, whole bacteria) from the 
intestine causes repeated infections, which lead to additional inflammation in the gut. In turn, 
this further damages the absorptive and barrier functions of the intestinal epithelium, trapping 
those who suffer with this condition in a cycle of infection, inflammation and malnutrition. 
 
At present, both EED itself and the role of increased gut permeability in EED (and in many of 
the other diseases highlighted above) are not well understood [1, 23, 24]. For this reason, the 
development of tools that can accurately monitor gut permeability and provide further 
information on its role in all of the above conditions is vitally important. Ideally, such devices 
should be minimally invasive and suitable for widespread deployment to allow large scale 
studies. Furthermore, if they are to be applicable to ailments that are prevalent in the 
developing world such as HIV and EED, these tools will also need to be low in cost. Importantly, 
systems of this sort could be useful not only in the investigation of the diseases above but also 
in providing a method for diagnosis, screening, and assessment of the efficacy of interventions. 
 
The most common technique that is currently used to directly assess gut permeability is the 
lactulose:mannitol (L:M) test [25]. Using this approach, patients receive an oral dose of the two 
sugars lactulose and mannitol, and urine samples are subsequently collected for up to six 
hours. The concentrations of the two sugars in urine are determined via liquid chromatography 
or mass spectrometry, and the L:M ratio provides an indication of the permeability of the 
intestine. A low L:M ratio indicates normal permeability, as lactulose is a large disaccharide 
molecule that is not absorbed by the healthy intestine (and, hence, is not found in urine) while 
mannitol is a smaller monosaccharide that passes the healthy intestinal barrier. An increase in 
the L:M ratio then indicates either increased permeability, impaired absorption, or a 
combination of the two. While the L:M test has been widely used to measure permeability, 
results are dependent on the proficiency of those carrying out the protocol as well as the level 
of diligence observed in dietary fasting (which is required prior to the test) and regular urine 
collection [25]. These problems are particularly acute when performing tests in infants, which 

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is desirable in the case of EED and, in some cases, coeliac disease. Moreover, in all cases 
the protocol is time consuming for both the patient and the care provider, and post-processing 
of urine samples is required in a laboratory, which can be challenging in developing world 
settings (where suitable infrastructure may not be available). 
 
Some alternative approaches based on similar methodologies are also available (see [2]), with 
one example involving the use of an orally administered polyethylene glycol (PEG) solution 
containing PEG molecules of varying molecular weights [1, 26]. Concentrations in urine are 
assessed in the same manner as described above, but more detailed permeability information 
is provided due to the wider range of molecular sizes being investigated (compared to just two 
in the L:M test). Nonetheless, this approach is prone to the same problems as L:M tests, 
suggesting that improved protocols will still be desirable. 
 
Endoscopic Biopsy and histology can also be used to assess the fragility of the intestinal 
epithelial barrier. However, this approach only provides an indirect, highly localised measure 
of the permeability and also represents an invasive procedure that is particularly undesirable 
in children and infants. 
 
Fluorescence spectroscopy may offer an alternative approach for the assessment of gut 
permeability, as it has previously been used in animal studies to quantify both intestinal closure 
and barrier function [27, 28]. In these investigations, intestinal permeability was assessed by 
feeding animals (rats and pigs) doses of fluorescent dextrans of varying molecular weights and 
then measuring the relative concentrations of these markers in the blood some time later. This 
was achieved using ex vivo measurements of blood samples and demonstrated that the 
passage of the fluorescent dextrans from the digestive system into the blood stream was 
dependent on both the state of intestinal closure and the size of the markers [27, 28]. 
 
Interestingly, it may be possible to translate this approach to non-invasive, in vivo use in 
humans through the use of transcutaneous detection of fluorescence (i.e. measurement of the 
fluorescence of the blood-borne markers through the skin). This method would entail a patient 
receiving an oral dose of a fluorescent contrast agent, with the concentration of that contrast 
agent in the bloodstream being measured in a transcutaneous arrangement using 
fluorescence spectroscopy. This approach – which would not require collection of urine or 
blood samples – would provide a direct measure of permeability and would be considerably 
less time consuming and labour intensive than the traditional L:M and PEG tests. Thus, it could 
be expected to offer considerable improvements in terms of both the reliability of the test and 
the experience of the patients. In addition, the devices used for fluorescence detection could 
be both low in cost and small in size, and only minimal post-processing would be required, 
making it possible to more quickly return test results to patients. 
 
As an aside, it is noteworthy here that a rate-limiting step for the entrance of substances into 
the intestine, and thus a factor in the quantification of gut permeability, is gastric emptying rate 
(GER). Gastric emptying is rarely considered, however its measurement would be highly 
complementary to that of gut permeability. In addition, it may have an effect on the ability to 
measure intestinal permeability using transcutaneous fluorescence spectroscopy. As such, 
control experiments will be performed here to determine the impact of GER on transcutaneous 
optical spectroscopy as a non-invasive tool for assessment of gut permeability. Interestingly, 
these control experiments may also serve to demonstrate the potential of transcutaneous 
fluorescence spectroscopy for non-invasive quantification of GER. 
 

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In the experiments proposed here we aim to test and validate transcutaneous fluorescence 
spectroscopy as a tool for the non-invasive quantification of gut permeability (and GER). This 
will entail testing two devices – a ‘mark I’ bench-top system and a miniaturised ‘mark II’ device 
suitable for larger scale deployment – along with four fluorescent contrast agents (fluorescein; 
indocyanine green, ICG; fluorescein isothiocyanate conjugated dextran, FITC-dextran; and 
FITC-conjugated PEG, FITC-PEG). These devices and contrast agents will be applied to 
patient studies with the key aims of validating transcutaneous spectroscopy, developing and 
optimising a fluorescence-based gut permeability assay (in terms of both the device and the 
protocol), and applying the optimised system to wide-ranging disease studies. The diseases 
investigated will include (but will not necessarily be limited to) coeliac disease, IBD, liver 
disease and HIV, as patients with these conditions will be accessible through Imperial College 
Healthcare NHS Trust. A longer term aim of this research (which is beyond the scope of this 
protocol) is to apply the fluorescence gut permeability assay to widespread studies of EED in 
low and middle income countries. Importantly, the successful development of this novel gut 
permeability sensor is likely to have a considerable impact in the broad field of gut health due 
to the opportunities it will offer for both non-invasive diagnostics and large-scale investigational 
studies. 
 
 
AIMS & OBJECTIVES 
 
This study has three overarching aims: 
 
1. to determine whether it is possible to monitor the permeability of the human gut 
using transcutaneous fluorescence spectroscopy of fluorescein, ICG, FITC-dextran 
and/or FITC-PEG; 
2. to develop, optimise and validate fluorescence-based permeability sensing via 
comparison to traditional gut permeability measurements (including PEG-based 
permeability assays and histological assessments) in healthy volunteers and 
positive controls (i.e. subjects with highly increased permeability such as untreated 
coeliac or IBD patients); 
3. to investigate a wide spectrum of gut permeabilities and to correlate transcutaneous 
permeability measurements to disease state in some or all of the conditions studied 
(which will include IBD, coeliac disease, HIV and fatty liver disease). 
 
 
MATERIALS & METHODS 
 
Instrumentation 
Two separate optical systems will be developed and used in this study. The first is a bench-
top device consisting of two laser sources for excitation of fluorescence, a spectrometer for 
detection, and a bifurcated optical fibre probe to allow interrogation of the fluorescence signal 
at the subjects’ skin (see Figure 1). This system is described in detail in Appendix A and is 
similar in design to the spectrometers reported in [29, 30]. Briefly, it entails two laser sources 
– at wavelengths of 488 nm and 785 nm – which permit excitation of fluorescence from 
fluorescein/FITC and ICG respectively. The fibre probe acts to deliver laser light to the subjects’ 
skin and to direct the excited fluorescence signal to the spectrometer. This allows for detection 
of the fluorescent dyes in the blood stream of the subjects. In order to ensure laser safety, the 
laser power at the distal end of the fibre probe will be limited. The allowable power levels will 
vary with the skin-probe distance, which will be tested in early experiments to determine the 

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optimum probe position. Thus, the optical powers will range from 63 µW (skin contact) to 
565 µW (maximum skin-probe separation of 2 mm – see Appendix A) at 488 nm and from 
93 µW (skin contact) to 836 µW (2 mm) at 785 nm. At these power levels the light output will 
be below the maximum permissible exposure for the skin (at the appropriate probe-skin 
separation) and will be eye-safe when the probe is held at a distance of >10 cm [31-33]. As 
the optical system is also contained within an interlocked, light-tight container, it can therefore 
be used by non-expert users in a clinical environment without the use of laser safety goggles 
and without risk of injury. To mitigate this risk even further, patients will be notified that they 
should not look directly at the tip of the fibre probe and will be supervised at all times during 
the experiments. 
 
 
 
Figure 1. Schematic diagram of the 'Mark I' bench-top optical system. All optical components are 
contained within a light-tight container that is interlocked to ensure the laser beam can only be 
manipulated by expert users. The optical power is attenuated using neutral density (ND) filters such that 
the output at the distal tip is below the maximum permissible exposure for skin at all times and is eye-
safe when held at a distance of >10 cm. In this configuration the device can be classified as a Class 1 
laser system and, hence, can be used by non-expert laser users in a clinical environment. The fibre 
probe consists of seven 200 µm diameter optical fibres, one for excitation (blue) and six for collection of 
fluorescence (yellow) – see insets at bottom right. At the distal tip of the probe the central excitation fibre 
is surrounded by the six collection fibres such that the ‘active area’ of the probe spans a diameter of 
approximately 600 µm. 
 
The second device used in this study will be a miniaturised version of the system described 
above. This device will use light emitting diodes (LEDs) for excitation and photodiodes (PDs) 
for detection of fluorescence (and for detection of the directly scattered laser light). It will entail 
a wireless, wearable tool that is secured in contact with the subjects’ skin during use. As above, 
the optical excitation powers will be limited in order that the system is eye-safe and skin-safe 
during use. The exact design of the ‘mark II’ device will be guided by the results collected with 
the bench-top (‘mark I’) system and the first stage of experimentation with the miniaturised 
system will entail validation against the bench-top version. All optical and electrical safety 
concerns will be factored into the design process, which is discussed in more detail in 
Appendix B. 

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As discussed in the Study Summary section, this research is inherently an investigation of a 
novel technique for the assessment of gut permeability rather than a study of a particular 
medical device. Indeed, the devices used in the study will be designed, amended and 
optimised based on the data collected throughout the investigation, with the goal of designing 
and validating an optimal approach for studies/measurements of gut permeability in large 
cohorts. 
 
Contrast agents 
As discussed above, four contrast agents will be used in these studies: fluorescein, ICG, FITC-
dextran and FITC-PEG. In Stage 1 of the experiments (see ‘Experimental protocols’ section 
below), either fluorescein or ICG will be administered to patients via intravenous injection. In 
Stages 2 and 3, combinations of fluorescein, ICG, FITC-dextran and/or FITC-PEG will be 
administered to patients orally (as a solution) with a variety of combinations tested (e.g. 
fluorescein + ICG, FITC-dextran/FITC-PEG + ICG, and fluorescein followed by FITC-
dextran/FITC-PEG). The order in which dye combinations will be tested in Stage 2 is described 
in the ‘Experimental Protocols’ section below. 
 
There are risks of side effects with all four of the proposed contrast agents, ranging from mild 
nausea in the least serious case through to anaphylaxis in the most serious. Importantly, 
however, patients participating in Stage 1 will only receive doses of contrast agent that have 
been prescribed clinically, meaning that these experiments will bring no additional risks 
(related to contrast agents). Furthermore, in Stages 2 and 3 all contrast agents will be delivered 
orally, which considerably reduces the risk of adverse events relative to intravenous use [34]. 
Nonetheless, in all cases, the dyes will be administered to subjects by trained healthcare 
professionals and all measurements will be carried out within a hospital environment. Thus, 
patients/volunteers will be monitored for signs of side effects at all times allowing rapid 
responses to any serious adverse events that occur. Doses will be limited to minimise the risk 
of complications while still allowing acceptable data to be collected (see summary of maximum 
dose levels in Table 1). The specific safety issues relating to each of the individual contrast 
agents are described below, and these are also explained in the participant information sheets 
so that subjects can make an informed choice about whether to take part in the study. 
 
Table 1. Maximum proposed doses for each of the fluorescent dyes to be used in this study. Maximal 
doses (for individual experiments) and the chosen administration routes are shown for each dye. In 
addition, relevant citations are given where further safety information can be found. Note that the total 
cumulative dose may be higher than the values shown in the Table in some instances, as some 
volunteers will be recruited for more than one spectroscopic gut permeability test. ICG – indocyanine 
green; FITC – fluorescein isothiocyanate; PEG – polyethylene glycol; IV – intravenous. 
 
Contrast agent 
Maximum dose 
Dose route 
Relevant citations 
Fluorescein 
500 mg (IV); 5 g (oral) 
IV and oral 
34-36, 38, 41, 44-47 
ICG 
5 mg/kg 
IV and oral 
50, 52, 54, 55 
FITC-dextran 
1 g 
Oral only 
56, 57, 60-62 
FITC-PEG 
1 g 
Oral only 
56, 57, 72-76 
 
Fluorescein Fluorescein is approved for medical use with intravenous delivery for 
ophthalmic angiography [35-37]. This is true despite the fact that adverse 
events – ranging from mild nausea through to anaphylaxis – are known to occur 
with varying frequencies. Mild adverse reactions (including mild nausea) are the 
most common, occurring in approximately 1/10 patients [35]. The frequency of 

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severe adverse events (including anaphylaxis) is much lower, at approximately 
1/2000 [34, 35]. In extremely rare cases, fatal allergic reactions have also been 
reported, accounting for approximately 1/200,000 patients [35]. Fluorescein is 
also often used ‘off-label’ with oral delivery – either as a solution or in the form 
of a tablet/capsule – for angiography [35, 36, 38-40], and this has been shown 
to be both safe and effective [34, 38, 41-44]. Indeed, it has been observed that 
minor adverse events are less common when administering fluorescein orally 
rather than intravenously [34, 44, 45] and reports of severe adverse events are 
extremely rare with oral fluorescein [34, 44] (two examples of anaphylaxis 
reported in response to oral fluorescein can be found in references [46, 47]). In 
addition, fluorescein has been further used ‘off-label’ for a variety of surgical 
and endoscopic imaging applications (e.g. [48, 49]). 
 
In the experiments proposed here, subjects will receive either an intravenous 
or oral dose of fluorescein depending on the experiment in which they are taking 
part. The maximal intravenous dose will not exceed 500 mg (typically 
administered as a 5 ml solution with a concentration of 100 mg/ml) as per the 
Medicines and Healthcare products Regulatory Agency (MHRA) guidelines [35, 
36]. Furthermore, the intravenous doses used will be those prescribed as part 
of the patients’ ophthalmic care and will not be increased or amended in any 
way as part of this study. In the case of oral delivery, a maximal dose of 5 g will 
be used. This is approximately double the maximal dose typically used in oral 
fluorescein angiography, however, it can be considered safe as complications 
are extremely rare with oral fluorescein delivery [34, 38, 41, 44, 45]. 
Furthermore, it will be necessary to test higher doses than those used for retinal 
imaging in order to determine the minimum oral doses that are detectable using 
a transcutaneous measurement protocol (i.e. lower doses will be detectable in 
the eye – due to its transparent nature – than through the skin). Once suitable 
minimum concentrations have been established, these will be used in all further 
experiments. 
 
ICG 
ICG is approved for medical use in solution using intravenous delivery for a 
variety of applications [50-53] and safe human use with oral delivery has also 
been reported (e.g. [54]). Complications are less common with ICG than they 
are with fluorescein (for intravenous delivery), with serious adverse events 
occurring with a rate of less than 1/10,000 and fatal allergic reactions having a 
frequency of below 1/330,000 [50]. In this investigation, subjects will receive 
either an intravenous or oral dose depending on the experiment in which they 
are taking part. The total dose delivered to a given patient will not exceed 5 mg 
of ICG per kg of body weight (mg/kg) for both intravenous and oral delivery. No 
significant toxic effects have been observed at this dose [52, 55] and it 
corresponds to the MHRA guidelines for intravenous use in adults [50]. Hence, 
it can also be expected to be a safe dose when administered orally. In addition, 
individual intravenous doses (i.e. those administered for a single experiment) 
will not typically exceed 0.5 mg/kg body weight, as per the MHRA guidelines 
[50]. Importantly, these dosage levels are also in agreement with doses used in 
investigational studies with oral dye delivery, in which this method of ICG 
administration was found to be safe (e.g. [54]). 
 

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FITC-dextran FITC-dextran is not yet approved for medical use, however, it has been applied 
to a limited number of in vivo clinical studies in humans using both intravenous 
[56] and oral [57] delivery. In addition, oral delivery of FITC-dextran has been 
used widely in animal experiments (e.g. [27, 28]). In human studies, total oral 
and intravenous doses of up to 1 g have been used (delivered as a 100 ml 
solution at 10 mg/ml orally and as a 10 ml solution at 100 mg/ml intravenously) 
[56, 57], and FITC-dextran has been shown to be non-toxic at up to 18 times 
this dose in rats [56]. Comparable oral doses have been used in animals, 
corresponding to 20-250 mg/kg in piglets [27, 28] and up to 2.5 g/kg in rats [28]. 
FITC-dextran is stable in solution and shows no evidence of degradation into 
smaller molecular weight species [56, 58]. This has been demonstrated both in 
vitro in solution [56, 58] and in vivo in rabbits after intravenous injection [58]. 
Furthermore, when applied orally in animals, FITC-dextran is detected in tact in 
the blood stream and the uptake into blood is dependent on both the molecular 
size and the state of intestinal closure [27, 28]. Similarly, after oral ingestion in 
humans, FITC-dextran is observed in tact in urine, with negligible free 
fluorescein detected [57]. Together, these results indicate that any degradation 
of FITC-dextran in the stomach and small intestine is minimal (although some 
breakdown of dextran into smaller molecular weight carbohydrates is expected 
to occur in the large intestine [59]). 
 
It is also noteworthy that dextran itself is approved for clinical use with 
intravenous delivery as a plasma expander [60]. However, serious anaphylactic 
reactions have been reported in response to dextran. These occur most 
frequently with dextran 70 (the largest molecular weight dextran used clinically) 
with rates estimated at between 1/2000 and 1/2700 patients [61, 62]. This 
corresponds to approximately twice the “background rate” of anaphylaxis 
reported in hospitalised patients [63]. Importantly, however, the doses of 
dextran used clinically are much higher than those proposed here – as a plasma 
expander, 15 g of dextran 70 are delivered in a 250 ml solution [60] and it is not 
uncommon that multiple sequential doses are used [61]. In addition, in these 
experiments, FITC-dextran will be delivered orally rather than intravenously, 
which will further mitigate the risk in three ways. Firstly, the rate of uptake into 
the blood stream will be much slower than with intravenous delivery (uptake is 
likely to occur over a timescale of several hours rather the 2-5 minutes 
recommended for intravenous delivery of dextran as a plasma expander [60]). 
Secondly, it is expected that there will be some digestion of large dextrans into 
smaller molecular weight carbohydrates by the bacteria in the large intestine 
[59], and it has been shown that smaller dextrans are less likely to induce 
anaphylaxis [61] (also see the use of dextran 1 to reduce allergic reactions to 
dextran 
40 
and 
70: 
https://www.nps.org.au/medical-info/medicine-
finder/promit-injection). Lastly, as discussed above for the specific case of 
fluorescein, serious adverse reactions to any potential allergens are much less 
likely with oral rather than intravenous delivery [34]. 
 
In the experiments proposed here FITC-dextran will be administered orally. In 
order to mitigate the risk, maximal doses of 1 g will be used. This corresponds 
to a dose that has previously been used safely in humans (using both 
intravenous and oral delivery) [56, 57] and is below the levels set out for oral 
delivery of fluorescein above. This dose is also well below the maximum 

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allowable dose of dextran, which is approved for use at considerably higher 
concentrations under certain circumstances [60] (see discussion above). Prior 
to administration, FITC-dextran solutions will be sterilised for clinical use via 
filtration through 0.2 µm filters, in accordance with the preclinical protocol 
reported in reference [56]. This will ensure that any heavy metal constituents or 
endotoxins are only present in very low concentrations that are suitable for 
human use [56]. As such, the risks presented by the oral delivery of FITC-
dextran have been minimised as far as possible. 
 
 
Figure 2. Molecular structures of FITC-dextran and FITC-PEG. (A) FITC-dextran. Asterisk (*) 
indicates that the location of the FITC binding is random and can occur at any hydroxyl group. (B) FITC-
PEG. Dotted red circles highlight the binding between the FITC moiety and the dextran/PEG moiety. 
The binding is similar in both cases indicating that the stability of the two molecules (at the highlighted 
binding site) in any given environment is likely to be comparable. 
 
FITC-PEG 
At present, FITC-PEG is not approved for clinical use and it has not yet been 
used in vivo in humans. Despite this, it has been used widely in research studies 
for both cellular (e.g. [64-69]) and animal imaging (e.g. [70]) experiments, where 
the PEG molecule is often used as a linker such that fluorescein-based 
fluorescence imaging can be combined with an additional functionality (e.g. 
thermal tumour ablation, selective binding, etc.) using a single molecule. In 
these studies, FITC-PEG has been shown to have a low toxicity in cells (in 
cases where it is not linked to an additional toxic moiety for therapeutic or other 
purposes) [64, 68-70]. Furthermore, PEG itself is used clinically [71] – for both 
intestinal permeability measurements [72, 73] and as a laxative [74-76] – and 
is known to be inert, non-toxic and stable (i.e. it is not broken down by gut 
bacteria, nor it is metabolised by GI tissues) [73]. Despite this, there are limited 
reports of serious adverse events (including anaphylaxis) being induced by 
PEG (even with oral delivery) [77-80], however, such reactions are extremely 
rare. In FITC-PEG, the binding between the FITC moiety and the PEG moiety 
is almost identical to the binding between FITC and dextran in FITC-dextran 
(see Figure 2). Thus, it can be expected that FITC-PEG will be stable within the 
GI tract in the same manner that FITC-dextran is (see above). As such, FITC-

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PEG can be considered safe for in vivo human use assuming that the 
concentrations of FITC and PEG are respectively kept below their maximum 
allowable doses. For this reason, we will use a maximum FITC-PEG dose of 
1 g, and this will be administered orally as a solution. This corresponds to the 
level set out for FITC-dextran above – which has previously been used safely 
in humans with both oral and intravenous administration – and is also below the 
maximum allowable dose of fluorescein. In addition, it is well below the 
maximum allowable dose of PEG, which is approved for use at considerably 
higher levels (e.g. 10-20 g as a laxative) [72-76]. As with FITC-dextran, prior to 
administration FITC-PEG solutions will be sterilised via filtration through 0.2 µm 
filters to allow safe clinical use. 
 
In experiments where two dyes (ICG + fluorescein or ICG + FITC-dextran/FITC-PEG) are 
administered simultaneously, the doses will be limited as described above. The aim of these 
experiments will be to determine whether both dyes can be detected simultaneously (through 
their differing excitation and emission profiles) and whether ratios of the fluorescence 
intensities of the two dyes can be used to provide a readout of permeability. 
 
In experiments where fluorescein and FITC-dextran/FITC-PEG are administered in sequence, 
the FITC-dextran/FITC-PEG dose will be taken into account when calculating the maximum 
fluorescein dose. In this way, both the total fluorescein dose and the FITC-dextran/FITC-PEG 
dose will be kept within the limits described above. These experiments will serve to investigate 
whether ratios of the fluorescein and FITC-dextran/FITC-PEG fluorescence intensities can 
provide a readout of permeability. In this case, administration of the dyes will be performed in 
sequence (rather than simultaneously) as the spectral properties of the contrast agents are 
very similar. 
 
Patient/subject selection 
We aim to recruit patients/subjects for this study from three groups as detailed below.  
 
Group 1 
Ophthalmology patients who are receiving an intravenous dose of either 
fluorescein or ICG as part of their routine ophthalmic care (e.g. as part of a 
fluorescence angiography examination). These patients will take part in early 
tests (Stage 1) determining whether it is possible to detect intravenous 
fluorescent 
dyes 
at 
known 
concentrations 
using 
a 
transcutaneous 
measurement protocol. 
 
Group 2 
Healthy subjects will be split into two groups (2a and 2b) dependent on the 
aspect of the study they participate in. 
 
                       2a: Healthy subjects with no known issues of increased gut permeability. These 
subjects will act as negative controls in all gut permeability studies. A subset of 
these volunteers will be asked to take two spectroscopic permeability tests – 
one with and one without a supplementary dose of a hyperosmotic solution, 
which acts to transiently increase intestinal permeability. This subset will 
provide proof-of-concept data prior to recruitment of patients in Group 3. 
 
                       2b: Healthy subjects with no known issues of increased gut permeability or gut 
motility. These subjects will participate in GER experiments, which will provide 
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as a non-invasive tool to assess gut permeability. Separate “healthy volunteer” 
sub-group will prevent the possibility of build-up of contrast agent. 
 
Group 3 
GI and non-GI patients who are expected to exhibit increased gut permeability 
(including, but not limited to: coeliac, IBD, HIV and liver disease patients). This 
group will represent the cases for the gut permeability study. Participants in this 
group who exhibit especially high intestinal permeabilities (as measured using 
control techniques such as PEG permeability assays or histology) will serve as 
positive controls. 
 
Eligible participants will be identified by the regular clinical teams in the relevant departments 
(HIV and Hepatology outpatient clinic, GI outpatient clinic, ophthalmology clinic). If a patient 
agrees to participate in the study their details will be passed on to the research fellow directly 
involved in the study. Participants will be asked to provide written informed consent. Healthy 
volunteers will be recruited from Imperial and hospital staff. 
 
Experimental protocols 
The experimental protocols will entail giving patients a dose of contrast agent as described 
above and then making transcutaneous fluorescence measurements for up to three hours by 
placing the optical probe in contact with the skin. In the case of the ‘mark I’ bench-top 
spectrometer, the tip of the fibre probe will be secured using a 3D-printed clamp such that it is 
either in contact with the skin or held up to 2 mm above the skin’s surface. A variety of 
measurement locations will be tested with this system in order to determine the most suitable 
for widespread deployment. The data from these studies will be used to guide the development 
of the casing for the miniaturised ‘mark II’ device (as well as the optical/mechanical/electrical 
design) such that it can be secured in the most appropriate body location. When using the 
miniaturised device, the entire system will be secured in place on the subject’s skin using a 
custom-designed 3D-printed casing (e.g. on the fingertip, arm, earlobe or other location, as 
determined by the bench-top measurements). In all cases, the skin at the measurement 
location will be cleaned/sterilised using 70% alcohol swabs and the probe/sensor will be 
cleaned in the same manner both before and after use. The exact experimental protocols will 
vary depending on the phase of the investigation and the subjects will also be asked to take 
some supplementary tests to provide data for validation of the spectroscopic gut permeability 
assay. Thus, the protocols are broken down into three stages (each with its own specific aims), 
which are described below and summarised in the flowchart presented in Figure 3. 
 
Stage 1 
In the first stage of the experiments we will recruit ophthalmology patients who 
are due to receive intravenous doses of fluorescein or ICG as part of scheduled 
angiography examinations. Measurements in these patients will serve to test 
whether it is possible to detect fluorescein/ICG in the blood using 
transcutaneous fluorescence spectroscopy at the known concentrations used 
for ophthalmic angiography. Experiments will be performed at the same time as 
the patient’s angiography meaning that they will require no additional hospital 
visits and no additional doses of fluorescent contrast agents (beyond that used 
for their eye examination). Before the fluorescent dye used for the angiography 
is administered (in accordance with the procedure recommended by the 
patient’s ophthalmologist), the spectrometer will be attached to the patient’s 
skin as described above. Background fluorescence data will be collected for 
five minutes before the patient receives their dose of fluorescein/ICG. 
Measurements will then continue for the duration of the angiography and for 

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some time afterwards (up to a maximum total experimental time of three hours, 
depending on the availability of the patient). Throughout the experiment 
fluorescence spectra will be recorded at regular intervals (e.g. every 30 s). 
These measurements will be carried out using the ‘mark I’ device in the first 
instance and they may also be repeated using the ‘mark II’ device as part of its 
validation. With the ‘mark I’ device, the probe-skin distance will be varied and a 
range of body locations will be tested as measurement sites in order to 
determine the optimum probe position/orientation. These preliminary tests will 
serve to optimise the protocols used in Stages 2 and 3. In the event that it is not 
possible to recruit a sufficient number of ophthalmology patients, we will also 
consider recruiting healthy volunteers for the Stage 1 intravenous injection 
protocol. If the transcutaneous detection of dyes is unsuccessful in the first 
instance then we will explore the use of more sensitive detectors as well as 
alternative fibre-optic probe designs. We will also consider increasing the 
concentration of contrast agents given to the subjects, but this will of course be 
conditional on further REC/HRA approval. 
 
 Stage 2 
The aim of the second phase of the experiments will be to validate 
transcutaneous fluorescence spectroscopy as a tool for the assessment of gut 
permeability. This requires two studies, and each will use different participants 
to prevent the possibility of build-up of contrast agent. Within the first study, we 
will recruit healthy volunteers (Group 2a) to take two spectroscopic gut 
permeability tests, the first under normal circumstances and the second with an 
additional dose of a hyperosmotic solution (which acts to transiently increase 
intestinal permeability). This will allow self-contained validation experiments to 
be performed in individuals (i.e. one healthy volunteer participating in two 
sequential fluorescence gut permeability tests, one with a dose of hyperosmotic 
solution and one without). In the first instance these volunteers will be given a 
combined oral dose of fluorescein and ICG as part of their spectroscopic gut 
permeability tests. This will permit measurements investigating the potential of 
each dye alone and the two dyes in combination to provide a readout of 
permeability (because the differing spectral properties will allow fluorescence to 
be recorded from both dyes simultaneously). If the fluorescein/ICG combination 
does not provide a suitable readout of permeability in the self-contained 
experiments in healthy volunteers, then we will recruit further healthy subjects 
and will investigate the use of alternative dye combinations and protocols (e.g. 
fluorescein followed by FITC-dextran/PEG or ICG combined with FITC-
dextran/PEG). The order in which dye combinations will be tested is illustrated 
in Figure 4, and we note that new subjects will be recruited for tests with new 
dye combinations. Hence, no subjects will be asked to take more than two 
spectroscopic gut permeability tests (in Stage 2) and, therefore, no subjects will 
receive doses of more than two of the four fluorescent contrast agents (in any 
Stage). 
 

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Figure 3. Flowchart describing experimental protocols and explaining the choice of contrast agents 
delivered at each stage of the investigation. The expected levels of recruitment are also given for each 
experimental step. Green arrows indicate progression through the protocol after successful 
experimental steps, red arrows indicate progression in the event of unsuccessful tests. Grp - group; IV 
- intravenous; ICG - indocyanine green. 
 
During the above validation experiments in healthy volunteers (Group 2a) it will 
also be necessary to test and optimise the concentrations of the fluorescent 
contrast agents used. To do this while also minimising each individual 
volunteer’s exposure to the dyes, we will begin with low doses of each dye and 
will only increase these doses if necessary (i.e. if the detected transcutaneous 
signals are too low to permit permeability sensing). For example, the first 
volunteer recruited to the study will receive a dose consisting of 500 mg 

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fluorescein and 1 mg/kg ICG (which are at the lower end of the values proposed 
in Table 1 and fall within the approved clinical limits for fluorescein and ICG). 
This volunteer will participate in two spectroscopic gut permeability tests (one 
without and one with a dose of hyperosmotic solution, as described above), 
even if no signal is detected in the initial experiment without the hyperosmotic 
solution. This is because increased permeability may have an effect on the dose 
required – i.e. at higher permeability it is likely that it will be possible to detect 
lower concentrations using the transcutaneous sensing setup (as more dye will 
leak from the gut into the blood stream). Based on the results from the first 
volunteer, the dose given to the second volunteer will be amended if necessary. 
If no (or very low) signal was observed (and if it cannot be reasonably expected 
that signal detection could be improved through changes to the spectrometer 
hardware) then the doses of fluorescein and/or ICG will be increased 
accordingly in a stepwise manner. If no signal was observed from either dye 
then the respective doses will be increased to 1 g fluorescein and/or 2 mg/kg 
ICG. The experiments in which the second volunteer participates will then be 
performed in an identical manner to those for the first volunteer. If these 
experiments also demonstrate a requirement for higher dye concentrations then 
in the following volunteers we will continue to increase the doses of fluorescein 
and/or ICG as necessary in steps of 500 mg and 1mg/kg respectively. This will 
continue until suitable concentrations are determined or until the maximum 
proposed dose levels are reached (see Table 1). Importantly, these dose 
escalation experiments will be performed in separate volunteers (i.e. they will 
be performed in a cohort not in an individual) so that individuals are not exposed 
to doses above those quoted in Table 1. Thus, in Stage 2, the maximum 
cumulative dose for any given dye will be twice that shown in Table 1, as 
volunteers will take no more than two spectroscopic gut permeability tests 
(Table 1 lists the maximum dose levels for individual tests). Group 2a and 2b 
will also be made up of different volunteers to prevent the possibility of contrast 
agent accumulation. Overall, these initial experiments will allow us to minimise 
the doses given to all future participants, thereby minimising the total exposure 
required throughout the study. 
 
Once the optimal dose of fluorescein has be determined, we will recruit healthy 
volunteers (Group 2b) to take a spectroscopic gut permeability test, using 
fluorescein as the contrast agent, and a paracetamol absorption test 
(standardised protocol for GER assessment of liquids). This will allow self-
contained validation experiments to be performed in individuals for GER (i.e. 
one healthy volunteer participating in two concurrent tests, one being a 
standardised method for GER measurement, and one being the transcutaneous 
fluorescence spectroscopy method). The paracetamol absorption method is a 
standardised protocol for GER assessment of liquids. As paracetamol is readily 
absorbed in the small intestine, and as GER is a rate-limiting step in substances 
entering the small intestine, the appearance of paracetamol in the blood 
provides an indirect measure of GER. Within this study, volunteers will arrive 
fasted having not consumed any food or drink (but water) in the last 12 hours. 
They will be given a combined oral dose of fluorescein (dose determined by the 
experiments discussed above) and paracetamol (1.5 g) within a 300-500 ml 
milk-based drink. Importantly, at these concentrations, all solutions are safe for 
human consumption. 1.5 g paracetamol is the recommend dose for 

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standardised GER measurements [81], and it represents 1.5x the usual dose 
used in home use. For each volunteer, blood samples (5 ml per sample) will be 
taken at regular intervals to allow subsequent quantification of the concentration 
of paracetamol in the blood (which will be used to determine GER). Samples 
will be taken over 4 hours. First, a baseline sample will be taken prior to 
consumption of the fluorescein/paracetamol dose. Then, for the first hour, blood 
samples will be taken every 10 minutes. Finally, for the remaining three hours, 
samples will be taken every 15 minutes. At the same time, measurements will 
also be made with the transcutaneous fluorescence spectrometer. The fibre 
probe will be attached to the arm of the participant and data will be collected at 
regular intervals (e.g. every 30 s). During the study, participants will be sat down 
and made comfortable. 
 
Measurement of GER effects will be achieved by comparing the fluorescence 
data to the standardised measure of GER (paracetamol absorption test). 
Specifically, data showing the rate of uptake of paracetamol into the blood will 
be compared against the transcutaneous fluorescence signal (rate of uptake of 
fluorescein). This will provide insight into the rate of substance entrance into the 
intestine, which will aid in the understanding and design of transcutaneous 
fluorescence spectroscopy as a measure of gut permeability. In addition, it will 
demonstrate whether transcutaneous fluorescence spectroscopy will also be 
suitable for non-invasive assessment of GER. 
 
 
Figure 4. Flowchart illustrating the order in which combinations of fluorescent contrast agents will be 
tested in the first step of Stage 2 (i.e. in healthy volunteers taking spectroscopic gut permeability tests 
both with and without a dose of hyperosmotic solution). Green arrows indicate experimental progression 
after succesful tests of a dye combination, red arrows indicate progression through the experiment in 
the event of unsuccessful tests. ICG – indocyanine green; FITC – fluorescein isothiocyanate; PEG – 
polyethylene glycol. 

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In the permeability study (Group 2a), if it is necessary to test FITC-dextran 
and/or FITC-PEG (i.e. as described above and in Figures 3 and 4), then the 
doses of these agents will also be minimised as far as possible. The initial doses 
will be determined based on the results obtained with fluorescein and the 
maximum proposed doses for FITC-dextran/PEG (1 g). If it is possible to detect 
fluorescein after oral ingestion at doses of below 1 g, then the initial dose of 
FITC-dextran/PEG will be set at the level determined for fluorescein. If the 
minimum detectable dose of fluorescein is greater than or equal to 1 g then all 
FITC-dextran/PEG experiments will be performed using a dose level of 1 g 
(which is the maximum proposed dose for these dyes, as set out in Table 1). In 
the case that an initial dose of below the maximum value of 1 g is used, then 
this will be increased in a stepwise manner (as described above for fluorescein 
and ICG) if necessary. For FITC-dextran/PEG, doses will be increased in steps 
of 100 mg. As above, these dose escalation experiments will be performed in a 
cohort and not in individual volunteers. Once suitable minimum doses have 
been established these will be used in all further experiments, thereby 
minimising the overall exposure. 
 
For all experiments (in all Stages of the protocol) and for all dye 
combinations/concentrations, measurements will be separated by a minimum 
of 24 hours so that it is possible to detect any adverse events that occur, even 
if they are delayed (by up to 24 hours). In the case that any serious adverse 
event occurs then a meeting of the Study Management Group will be triggered 
and all safety concerns will be discussed before any further experiments are 
performed (see Safety Monitoring section below for further details). 
 
Once we have determined a suitable measurement protocol and combination 
(and concentration) of contrast agents in healthy volunteers (Group 2a), we will 
seek to recruit patients with GI (or other) conditions who are expected to exhibit 
particularly high intestinal permeabilities (e.g. untreated coeliac or IBD 
patients). These patients (Group 3) will serve as positive controls, and the 
technique will be further validated by comparing the data collected in patients 
with that collected in healthy volunteers (without doses of hyperosmotic 
solutions).  
 
Throughout Stage 2, all subjects will be given oral doses of the fluorescent 
contrast agents and transcutaneous fluorescence measurements will be made 
using the ‘mark I’ bench-top device. 
 
 
In all cases, once patients/volunteers have agreed to take part in the study, a 
time will be arranged for them to come into the hospital for measurements to be 
taken. On the day of their study visit, the subjects will first be asked a series of 
questions regarding their general details, medical history and current diet 
(including details of any medication they are taking and their current alcohol 
intake). Measurements will also be made of the subjects’ height, weight and 
waist circumference, and we will ask for consent to access their medical 
records. At this point we will also ask female participants to take a pregnancy 
test and if this returns a positive result then they will be excluded from the study. 
Once the preliminary questions and measurements are complete, the optical 

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probe will be secured in contact with the skin and the subject will receive an oral 
dose of fluorescein, ICG, FITC-dextran, FITC-PEG or a combination thereof 
(with the doses limited as described in the ‘Contrast agents’ section). Where 
GER is measured (Group 2b), only fluorescein will be used as the contrast 
agent. Transcutaneous fluorescence measurements will be recorded at regular 
intervals (e.g. every 30 seconds) for up to 3 hours (although longer experiments 
may be considered in patients who are expected to exhibit slow intestinal transit 
and/or slow gastric emptying), beginning five minutes before the contrast agent 
is administered in order to allow collection of a baseline (for Group 2b, the time-
course detailed above will be used). During the experiment, two 5 ml blood 
samples will also be taken – one prior to administration of the contrast agent 
and one at the point at which the peak fluorescence intensity is detected – in 
order to allow correlation of the fluorescence measurements with the blood 
concentration of the fluorophores. For the same purpose, two urine samples will 
also be collected – one before administration of the contrast agent and one at 
the end of the experiment. The blood/urine concentrations will be measured 
using a laboratory-based spectrofluorometer (as described in [27]) after the 
transcutaneous fluorescence procedure is complete. Some of the collected 
blood and urine may also be used to perform other tests, for example to assess 
the degree of bacterial translocation. 
 
During the GER experiments (Group 2b), the blood sample regime detailed 
above (page 22) will be used, which is in line with standardised methods for 
paracetamol absorption testing. These blood samples will be analysed for 
paracetamol concentration by North West London Pathology (Imperial College 
Healthcare NHS trust), and no additional blood (or urine) samples will be taken 
for the purposes of assessing concentration of fluorescent dyes or bacterial 
translocation. 
 
 
Following the spectroscopic gut permeability tests, Group 2a/3 subjects will be 
asked to make two further contributions to the study. Firstly, they will be asked 
to provide one or more fresh stool samples within approximately 48 hours of 
their study visit. These will be analysed to determine the concentration of 
fluorescent dye in the stool (which will provide an indication of intestinal transit 
time) and may also be assessed with the aim of identifying and quantifying the 
microbiota. Secondly, Group 2a/3 subjects will be asked to take a PEG 
permeability test, which will act as a ‘gold standard’ measurement against which 
we will compare our spectroscopic permeability readings. Subjects will be given 
a specific collection kit that will allow them to take the PEG permeability test at 
home at a convenient time. The test entails drinking a 250 ml aqueous solution 
of PEG molecules of varying molecular masses and then collecting urine for the 
following six hours. The urine sample is then shipped to a laboratory for analysis 
using prepaid packaging provided by the research team. 
 
 
In patients who have had or are due to have an intestinal biopsy as part of their 
clinical care, we will ask for consent to use any tissue from the biopsy that is 
surplus to diagnostic requirements for further analysis in our study. This will 
allow correlation of the spectroscopic gut permeability test with histological 
measurements of epithelial damage or permeability (e.g. quantification of the 
efficacy of the tight junctions via Ussing Chamber measurements). 

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Finally, as discussed above, in the subset of Group 2a (healthy subjects) 
recruited at the beginning of Stage 2, we will ask the volunteers to take the 
spectroscopic gut permeability test twice. In this group, the first fluorescence 
gut permeability test will be performed as described above. In the second test, 
however, the volunteers will receive a dose of the chosen fluorescent contrast 
agent(s) in combination with a hyperosmotic solution that will act to transiently 
increase their gut permeability (i.e. in a similar manner to that described in 
references [57, 82]). Thus, by comparing the results in these subjects with and 
without the hyperosmotic solution, we will be able to validate the capability of 
the spectroscopic test to monitor changes in intestinal permeability. 
Interestingly, this validation will be possible without the requirement for 
additional ‘gold standard’ measurements (such as PEG permeability tests or 
histopathology, as discussed above) as each subject will act as their own 
internal control. A range of hyperosmotic solutions will be investigated, including 
sucrose, glucose, glycerol and sodium chloride, with maximum doses of 60 g, 
30 g, 15 g and 5 g (in 100 ml of water) respectively. Importantly, at these 
concentrations, all solutions are safe for human consumption (all are widely 
used in the food industry – for example, a 500 ml bottle of Coke contains 
approximately 50 g of sugar) and can be expected to instigate temporary 
alterations in intestinal permeability [57, 82]. These self-contained experiments 
in individual healthy volunteers will be performed before measurements in 
patients in order to provide a preliminary validation of the technique (and to 
determine the most valuable experimental protocol and combination of contrast 
agents). 
 
 
Overall, the aim of Stage 2 is to develop, optimise and validate the 
spectroscopic assay proposed in this protocol as a readout of gut permeability. 
This will involve determining the optimum combination and dose of contrast 
agents, controlling for GER effects, and investigating data analysis/processing 
approaches to ascertain which offer the optimal permeability readouts. This will 
be achieved by testing a range of contrast agent combinations and analysis 
protocols, and assessment of GER effects will be achieved by comparing 
fluorescein in relation to a standardised measure of GER (paracetamol 
absorption test). Measurements will first be performed in healthy volunteers 
taking two spectroscopic gut permeability tests (one with and one without a 
dose of hyperosmotic solution) and later in patients expected to exhibit 
increased intestinal permeability. In both cases, the data acquired will be 
compared to one or more of the ‘gold standard’ measurements discussed 
above. 
 
Stage 3 
Based on the data collected in Stage 2, we will design and develop an 
optimised, wearable device (‘mark II’) – along with an optimised experimental 
protocol – suitable for larger scale studies. This device and protocol will then be 
applied to disease studies aimed at: (i) demonstrating the capability of the 
optimised spectroscopic gut permeability assay to accurately monitor a wide 
range of permeabilities; and (ii) correlating spectroscopic measures of 
permeability to disease state (via comparison to the ‘gold standard’ markers 
discussed above). The experimental protocol used will be identical to that 
described for Stage 2, but the use of the ‘mark II’ device and the optimised 

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procedure (which will most likely entail a much shorter measurement duration) 
will allow for measurements in considerably larger cohorts. We will aim to recruit 
patients expected to exhibit increased gut permeability with a variety of 
diseases. This will include (but will not necessarily be limited to) inflammatory 
bowel diseases (such as Crohn’s disease and colitis), coeliac disease, fatty liver 
disease, and HIV. In cases where patients receive a treatment/therapy that 
begins after their planned spectroscopic gut permeability test, we will also seek 
consent to make multiple longitudinal measurements to allow us to determine 
whether this approach can be used to monitor the impacts of interventions. At 
this stage of the experimental program it is possible that we will have the 
opportunity to extend the level of recruitment beyond that set out in the Sample 
Size & Statistical Analysis section. If this is the case then we will seek further 
ethical approval – via an amendment to this protocol – before recruiting 
additional participants. Overall, the experiments performed in Stage 3 will serve 
to demonstrate the utility of the spectroscopic gut permeability assay in a wide 
array of prevalent disorders. 
 
Schedule of procedures 
As described above, participants will take part in a series of procedures as part of this study, 
with some carried out in the hospital by the study team and others undertaken at home by the 
subjects/patients themselves. The procedures will vary depending on the stage of the 
investigation and the patient/subject group. Thus, the table below shows and describes the 
procedures that will be performed for each patient group. 
 
Table 2. Description of study procedures. The column labelled ‘Group’ indicates which patient groups (see 
‘Patient/subject selection’ above) will participate in each procedure. 1 – Ophthalmology patients; 2 – healthy 
volunteers; 3 – GI and non-GI patients with expected increased gut permeability. 
 
PROCEDURE 
DESCRIPTION 
VISIT 
GROUP 
Informed consent 
 
The Study Doctor will discuss the Patient Information Sheet 
and Informed Consent Form with the patient and will answer 
any questions. Once the patient feels satisfied that their 
questions have been answered and feels certain that they 
want to join the study, the doctor will ask them to sign the 
Informed Consent Form. At this point we will also arrange a 
time for the patient to come in and participate in the study 
(the study visit). 
 
Consultation 
visit (prior to 
study) 
1, 2a, 2b, 
3 
Inclusion/exclusion 
criteria 
The Study Doctor will go through the inclusion and exclusion 
criteria to ensure that the patient is eligible to join the study 
by asking questions regarding medical history.  
 
Consultation 
visit (prior to 
study) 
1, 2a, 2b, 
3 
Pregnancy test 
Before taking any further part in the study, female volunteers 
will be asked to take a pregnancy test. If this returns a 
positive result then they will be excluded from the study. This 
step will only be performed in Stages 2 and 3. In Stage 1, 
inclusion of pregnant women will be at the discretion of the 
patient’s ophthalmologist. 
 
Study visit 
2a, 2b, 3 
General, medical 
and dietary 
questions; 
Subjects will be asked a few questions about their general 
details and medical history, including any medication they 
are taking. Height, weight and waist circumference will be 
Study visit 
1, 2a, 2b, 
3 

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PROCEDURE 
DESCRIPTION 
VISIT 
GROUP 
measurements of 
height, weight, etc. 
 
measured. Subjects will also be asked some questions 
about their diet, including current alcohol intake. 
 
Consent to use 
tissue from 
intestinal biopsy 
If patients in group 3 have had or are due to have an 
intestinal biopsy as part of their clinical care, we will ask for 
their consent to use any surplus tissue from the biopsy in our 
study. This will allow us to correlate the results of the 
spectroscopic gut permeability test with histological markers 
of tissue damage or increased intestinal permeability. 
 
Study visit 
3 
Intravenous 
injection of 
contrast agent: 
fluorescein or ICG 
Ophthalmology patients will receive an injection of either 
fluorescein or ICG, as prescribed by their ophthalmologist. 
The dye and dose used will be exactly the same as for their 
normal angiography examination. 
 
Study visit 
1 
Oral administration 
of contrast agent: 
fluorescein, ICG, 
FITC-dextran 
and/or FITC-PEG 
Group 2a and 3 volunteers will receive an oral dose of one 
or 
more 
fluorescent 
dyes 
(fluorescein, 
ICG, FITC-
dextran and/or FITC-PEG). This will be administered as a 
single solution of approximately 300 ml (similar to the size of 
a can of soft drink) for the subject to drink.  
 
Study visit 
2a, 2b, 3 
Group 2b volunteers will receive an oral dose of fluorescein. 
This will be administered as a single solution in a 300-500 ml 
milk-based drink that also contains 1.5 g paracetamol. 
 
Spectroscopic gut 
permeability test 
We will attach a small device (spectrometer) to the arm or 
fingertip of the subject. This device will be used to make 
measurements of the amount of contrast agent in the blood 
stream, through the skin. The device is comfortable to wear 
and will not break, damage or hurt the skin in any way. 
 
Study visit 
1, 2a, 2b, 
3 
Oral dose of 
hyperosmotic 
solution 
A subset of the healthy volunteers will receive an oral dose 
of a hyperosmotic solution (which will act to temporarily 
increase their gut permeability) as part of a second 
spectroscopic gut permeability test. This will take place at a 
second study visit. Other than the dose of the hyperosmotic 
solution (which will consist of sucrose, glucose, glycerol or 
sodium chloride), the procedure will be identical to the 
volunteers’ first spectroscopic gut permeability test. 
 
Second 
study visit 
2a 
Blood samples 
Group 2a: We will take two 5 ml blood samples during the 
tests (equivalent to about one teaspoon of blood for each 
sample). The first will be taken before the measurements 
begin 
and 
the second will 
be 
taken 
during 
the 
measurements. 
 
Study visit 
2a, 2b, 3 
Group 2b:  For each participant, we will take nineteen blood 
samples (up to 5 ml) over the course of 4 hours. One sample 
will be taken at baseline, followed by one every 10 minutes 
for the first hour, then one every 15 minutes for the final 
3 hours. 
 

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PROCEDURE 
DESCRIPTION 
VISIT 
GROUP 
Urine samples 
We will ask participants to provide two urine samples. The 
first will be collected before the measurements begin and the 
second will be collected at the end of the measurements. 
Study visit 
2a, 3 
Adverse events 
Participants will be asked if they are undergoing or have 
experienced any adverse events (such as any problems, 
symptoms or discomforts). 
 
During and 
after study 
visit 
1, 2a, 2b, 
3 
Stool sample 
 
Within 48 hours of the Spectroscopic gut permeability test 
we will ask subjects to provide one or more fresh stool 
samples, which will be used for further analysis. 
 
2 days after 
study visit 
2a, 3 
PEG gut 
permeability test 
 
Finally, subjects will be asked to take a PEG permeability 
test sometime in the week following their study visit. This can 
be carried out at home at the convenience of the participant, 
and they can administer the test themselves. We will provide 
a collection kit to perform the PEG permeability test, which 
will involve drinking a solution and then collecting urine for 
the following 6 hours. Subjects will then post the collected 
urine sample to a laboratory for analysis using prepaid 
packaging. We will ask that subjects try to perform the PEG 
permeability test within 1 week of their study visit. 
 
2+ days 
after study 
visit 
2a, 3 
 
Safety Monitoring 
The Study Management Group will also act as a Safety Monitoring Committee for all 
experiments proposed in this protocol. The group is well suited for this purpose as it contains 
experts in intestinal health, liver health, ocular health (where fluorescein and ICG are widely 
used) and laser safety. The group will meet on a monthly basis to review experimental results 
and, when necessary, to take decisions as to whether to advance to the next experimental 
stage/step (as described in Figures 3 and 4). In these meetings, any and all safety concerns 
will also be discussed. If any non-serious adverse events (see definitions in Adverse Events 
section below) have been reported then these will be discussed in detail and decisions will be 
taken with regards to the specific experimental procedures used in the case of the adverse 
events – i.e. the committee will decide whether a particular procedure (e.g. a particular dye 
combination/concentration or a particular laser power / measurement location) presents an 
unexpected and unacceptable risk. In this situation the committee will discuss whether it is 
sensible to move on to an alternative experimental protocol, to perform further measurements 
using the same protocol, or to terminate the study completely (i.e. as per the flowcharts shown 
in Figures 3 and 4). 
 
In the case that a serious adverse event is reported, then all measurements/experiments will 
be temporarily postponed with immediate effect and an exceptional meeting of the Study 
Management Group will be triggered. At this meeting the committee will discuss the serious 
adverse event in detail (as well as any other safety concerns that have arisen since the 
previous meeting) and will determine whether it is safe to proceed with further experiments or 
whether termination of the study is necessary. 
 
Patient recruitment 
Potential participants will be approached by members of their healthcare/clinical team. This 
will either be in the outpatient department or prior to their appointment by telephone. For those 

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patients approached on the phone, patient information sheets will be sent to them in the post 
prior to their outpatient appointment. All patients who agree to see the study team will then be 
provided with an information sheet (if they were not already sent one) and given an opportunity 
to discuss their involvement in the study after their clinic appointment. Informed consent will 
be taken by trained (GCP accredited) clinical members of the study team after a minimum of 
24 hours has elapsed from the time at which the study information was provided. 
 
Healthy volunteers will be recruited from Imperial College and St. Mary’s Hospital staff. 
Potential healthy volunteers will be approached in person by members of the research team 
or recruited via advertisements, which will be displayed around Imperial College campuses 
and circulated to staff/students via e-mail (attached separately). Study information will then be 
provided as described above and informed consent will be taken by trained (GCP accredited) 
clinical members of the study team (a minimum of 24 hours after the study information was 
provided). 
 
Subjects taking part in Stages 2 and 3 of this research program will be offered a fixed financial 
incentive of £20 to encourage participation in the study. Subjects will receive this fee upon 
completion of all aspects of the study in which they agreed to take part. Transport costs will 
not be reimbursed due to the limited level of funding. Volunteers participating in Stage 1 of the 
study will not be offered financial incentives as the impact on subjects is much smaller in Stage 
1 than it is in Stages 2 and 3. This information will be made available to potential participants 
in the participant information sheets. 
 
 
INCLUSION CRITERIA 
 
 
• 
Ability to give informed consent 
• 
Aged 18 years or above 
• 
No evidence of prior adverse reactions to fluorescein, ICG, dextran or PEG (dependent 
on experiment) 
• 
No evidence of prior adverse reactions to iodine (for ICG experiments only) 
• 
For healthy volunteers: healthy with no active GI/liver disease (or other condition in 
which increased gut permeability is expected, e.g. HIV) and no antibiotics taken within 
the previous four weeks. 
• 
For cases: exhibiting symptoms of GI, liver or other diseases (e.g. HIV) in which 
increased intestinal permeability is expected. 
• 
For ophthalmology patients recruited in Stage 1: healthy (i.e. as described above 
for healthy volunteers) and prescribed to have an ophthalmic angiography with an 
intravenous injection of either fluorescein or ICG. 
 
 
EXCLUSION CRITERIA 
 
• 
Unable to give informed consent 
• 
Aged <18 years 
• 
Previous adverse reaction to fluorescein, ICG, dextran or PEG (Group 1, 2a, 3) 
• 
Previous adverse reaction to fluorescein (Group 2b) 
• 
Known allergy to iodine (for ICG experiments only) 
• 
Pregnancy (in Stage 1 this will be at the discretion of the patient’s ophthalmologist) 

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• 
Breastfeeding (in Stage 1 this will be at the discretion of the ophthalmologist) 
• 
Known allergy to paracetamol (Group 2b only) 
• 
Known allergy to ingredients in milk-based drink (i.e. dairy, nuts, gluten) used in gastric 
emptying experiments (Group 2b only) 
 
 
WITHDRAWAL OF SUBJECTS 
 
Subjects are free to withdraw consent for inclusion in the study at any time and, where 
requested, their samples/data will be removed from further study and destroyed.   
 
 
ADVERSE EVENTS 
 
Definitions 
 
Adverse event (AE): any untoward medical occurrence in a patient or clinical study subject. 
 
Serious adverse event (SAE): any untoward and unexpected medical occurrence or effect 
that: 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject was at risk of death 
at the time of the event; it does not refer to an event which hypothetically might 
have caused death if it were more severe 
• 
Requires 
hospitalisation, 
or 
prolongation 
of 
existing 
inpatients’ 
hospitalisation 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations. Important AEs that are not immediately life threatening or do not result in death or 
hospitalisation but may jeopardise the subject or may require intervention to prevent one of the 
other outcomes listed in the definition above, should also be considered serious. 
 
Reporting procedures 
All AEs should be reported. Depending on the nature of the event the reporting procedures 
below should be followed. Any questions concerning adverse event reporting should be 
directed to the Chief Investigator in the first instance. 
 
Non-serious AEs 
All such events, whether expected or not, should be recorded. 
 
Serious AEs 
An SAE form should be completed and sent to the Chief Investigator within 24 hours. However, 
relapse and death due to a pre-existing condition, and hospitalisations for elective treatment 
of a pre-existing condition do not need reporting as SAEs. 
 
All SAEs should be reported to the London – Bromley REC where in the opinion of the Chief 
Investigator, the event was: 

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• 
‘related’, i.e. resulted from the administration of any of the research procedures; 
and 
• 
‘unexpected’, i.e. an event that is not listed in the protocol as an expected 
occurrence. 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the NRES (National Research Ethics Service) 
SAE form for non-IMP (investigational medicinal product) studies. The Chief Investigator must 
also notify the Sponsor of all SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office. 
 
Contact details for reporting SAEs 
 
Sponsor 
Imperial 
College 
Joint 
Research 
Compliance 
Office 
– 
jrco@imperial.ac.uk. 
 
Chief Investigator 
Dr Alex Thompson – alex.thompson08@imperial.ac.uk, +44 (0) 20 3312 
5035. 
 
 
SAMPLE SIZE & STATISTICAL ANALYSIS 
 
We aim to recruit 70 subjects to this study. We expect this to be an achievable number as we 
will have access to patients through the Endoscopy unit and the Hepatology and HIV clinics at 
St Mary’s Hospital as well as the Ophthalmology clinic at Western Eye Hospital. 
 
This will include a total of 40 subjects with no suspected intestinal health issues. This subset 
will be made up of ophthalmology patients undergoing fluorescein or ICG angiography and 
healthy volunteers. We will aim for 5 of these patients to receive intravenous doses of 
fluorescent contrast agents (ophthalmology patients) and for the remaining 35 (healthy 
volunteers) to be given oral doses (Group 2a – 15 volunteers; Group 2b – 20 volunteers). The 
measurements on subjects receiving intravenous dyes will serve to allow a simple test as to 
whether the fluorescence signal can be detected in a transcutaneous arrangement. The 
measurements on subjects receiving oral doses will act as the healthy control measurements 
in which intestinal permeability is expected to be normal. As discussed above, some healthy 
volunteers in Group 2a will take the test twice in order to assess repeatability or to validate the 
ability to detect changes in permeability (with doses of hyperosmotic solutions used to induce 
temporary increased permeability). 
 
A further 30 patients will be recruited who are expected to show increased intestinal 
permeability. These patients will have either GI conditions or non-GI conditions such as HIV 
and liver disease for which increased intestinal permeability is expected. 
 
The study will serve to answer a number of clinical questions including whether transcutaneous 
fluorescent signals can be detected after intravenous and oral administration of contrast agents 
and whether these signals correlate to traditional (either direct or indirect) measures of gut 
permeability (i.e. PEG-based permeability assays or histological assessments). The most 
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can be used to differentiate between subjects with normal intestinal permeability and those 
with increased permeability. Therefore, we estimated the number of patients required to 
answer this question with a statistical power of over 80% and an alpha value of below 0.05. 
This calculation requires knowledge of the mean values and standard deviations that will be 
obtained using the fluorescence permeability assay. As these are currently unknown (because 
this is a new test), we used previously reported L:M (lactulose:mannitol) ratios to estimate the 
power and alpha values. L:M ratios are likely to provide a similar readout to the fluorescence 
permeability assay as we will be investigating differences (ratios) between the fluorescence 
intensities of small (fluorescein/ICG) and large (FITC-dextran/FITC-PEG) fluorophores. Thus, 
the L:M ratio (which measures the relative urinary recovery of one large and one small sugar 
molecule) can be considered analogous to the fluorescence assay, and previously reported 
L:M ratios should serve as a suitable substitute in these calculations. 
 
In healthy patients with normal intestinal permeability, L:M ratios are typically on the order of 
0.01-0.02 [83, 84]. In patients with coeliac disease and Crohn’s disease, increased L:M ratios 
have been reported, corresponding to 0.105 [84] and 0.085 [83] respectively. Considerable 
intra- and inter-patient variation is observed in both healthy volunteers and patients with 
increased intestinal permeability, with standard deviations of up to approximately 100% of the 
mean values having been reported [83, 84]. 
 
Using the patient numbers and L:M ratios above, we can calculate the statistical power for a 
comparison of the means of two samples (i.e. healthy volunteers vs. patients with increased 
permeability). With 45 patients (15 healthy, 30 increased permeability), following the 
calculation described at http://powerandsamplesize.com/Calculators/Compare-2-Means/2-
Sample-Equality, we find that an alpha value of 0.05 and a statistical power of above 
approximately 80% can be obtained even when using the highest observed standard 
deviations. For example, using a standard deviation of 0.085 for both the normal permeability 
and increased permeability cohorts, with L:M ratios of 0.021 and 0.085 respectively (i.e. as 
reported by Andre et al. [83], with standard deviations for both cohorts estimated as 100% of 
the increased permeability mean), we obtain an alpha value of 0.05 and a statistical power of 
79.2%. Using lower standard deviations or greater differences between the two means 
provides even higher statistical powers. This represents acceptable statistical significance and 
indicates that with 45 patients (assuming the means and standard deviations above) we would 
be able to confidently reject the null hypothesis that the mean permeabilities of the healthy and 
increased permeability cohorts are in agreement (H0: ma – mb = 0) – i.e. we would be able to 
confirm that the fluorescence-based gut permeability assay was capable of detecting changes 
in intestinal permeability. 
 
Of course, the actual mean values and standard deviations observed when using the 
fluorescence-based permeability assay will only be determined once the experiments 
commence. However, the above estimates represent acceptable statistical parameters, 
suggesting that the study will be capable of producing statistically significant results based on 
the proposed level of recruitment (assuming that the mean values and standard deviations 
obtained with the fluorescence-based assay are comparable to those reported in L:M tests). 
Furthermore, the patients recruited to this study as cases will naturally exhibit a range of 
permeabilities and it may be possible to use the fluorescence assay to differentiate between 
subgroups within the increased permeability cohort based on a variety of factors (e.g. disease 
type, disease state, treatment duration, etc.). While these are important research questions, 
we have based the above calculation of statistical power solely on the experiments designed 
to provide initial validation of the technique, as this is the key aim at this stage of the research 

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program. If differences are observed between subgroups that are not statistically significant at 
this level of recruitment then we will later seek to recruit additional patients (through an 
amendment to this protocol) to examine these phenomena in detail. Similarly, if it is not 
possible to obtain statistically significant results at the proposed level of recruitment because 
it is necessary to test multiple different dye combinations (i.e. as shown in Figure 4) before a 
suitable permeability readout is obtained, then we will also seek ethical approval to recruit 
additional subjects through an amendment to this protocol. In the first instance, however, 50 
subjects represents a level of recruitment that we believe is feasible within the timescale of the 
funding for this research program and that has the potential to provide statistically significant 
findings as described above. 
 
For GER experiments, the aim of the measurements is to assess the impact of GER on the 
ability to measure intestinal permeability and to perform a preliminary validation of 
transcutaneous fluorescence spectroscopy as a tool to quantify GER (by comparing collected 
fluorescence data against information from paracetamol absorption tests). As above, the mean 
values and standard deviations that will be obtained are unknown (as this is a new test), which 
means that suitable data are unavailable for power calculations. Therefore, a sample size of 
20 healthy volunteers for the GER experiments (Group 2b) was chosen based on typical 
samples sizes used in previous validation studies for alternative techniques (e.g. see the 
review of paracetamol absorption test validation studies presented by Willems et al. [81]). 
 
Statistical analysis will be performed using Matlab, and professional statistical advice will be 
sought where necessary. 
 
DATA COLLECTION, DATA HANDLING & RECORD KEEPING 
 
Explicit consent for access to medical records by members of the research team will be gained. 
Transfer on magnetic/optical media or networks will only be in encrypted form, according to 
local NHS ICT protocols. Where data is stored on NHS or Imperial College computers, 
appropriate access controls will be in place to ensure that access to confidential research 
information is restricted to those who need it. Paper records (consent forms etc.) will be stored 
securely on NHS premises. This will be within a locked filing cabinet or cupboard in a locked 
office to which only the senior research team has access. 
 
The Data Protection Act and Caldicott principles will be adhered to at all times. 
 
Data will be pseudo-anonymised as soon as possible. Data recorded on the case record form 
will be identified by a unique reference number. This will only be linked to the individual patient 
separately, in a secure database held by the recruiting clinicians. Samples, case record forms 
and other trial documentation will be labelled only with this unique identifier. 
 
Identifiable patient data will only be stored on secure computers which may only be accessed 
by the clinicians involved in the patients' clinical care. A unique identifying numerical code – 
which is distinct from the NHS number or hospital record number – will be assigned to each 
record. This unique identifier will be used for all research data stored on investigators’ 
computers. This pseudo-anonymised data will be kept on NHS and University computers. Such 
data will be encrypted to the local ICT requirements. 
 
Only members of the research team who hold a relevant NHS Trust contract will have access 
to the medical records of those who agree to participate. Explicit consent for this access will 

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be sought from each participant on the consent form. Information gleaned from such access 
will remain entirely confidential, and will only be recorded anonymously in study records. 
 
It is Imperial College policy that all data relating to research, including consent forms, are kept 
for 10 years. 
 
 
SAMPLE STORAGE 
 
Any samples collected from patients will be handled and processed in line with relevant 
biosafety regulations. In the case of samples collected from HIV-infected patients, all 
processing will be undertaken within a category 3 laboratory. 
 
All samples (except those from Group 2b, where blood samples will be processed by an NHS 
pathology laboratory and then destroyed) will be registered and stored as a sub-collection of 
Imperial’s Tissue Bank. This Tissue Bank fully conforms to HTA regulation. All freezers in the 
unit are constantly monitored by T-Scan alarm systems and are fully secured. Access to 
samples is by permission of the study CI. Freezers are locked and are kept within rooms with 
pin code entry systems. 
 
At the end of the research, samples where consent allows for future research use will be 
transferred into the Biobank, which operates under the licence of Imperial College. 
 
 
STOPPING/DISCONTINUATION RULES 
 
The study is due to continue for four years. It is anticipated that healthy subject and 
ophthalmology patient measurements will be undertaken in the first year, while measurements 
on GI patients and other non-GI patients with increased gut permeability will continue for the 
duration of the study. There are no specific stopping rules as this is primarily a feasibility study 
investigating the novel gut permeability assay. 
 
 
RESEARCH GOVERNANCE, MONITORING, ETHICS AND R&D APPROVAL 
 
The study will be conducted in compliance with the Research Governance Framework for 
Health and Social Care and Good Clinical Practice. The study will be conducted in accordance 
with the approvals of the Research Ethics Committee and the Joint Research Compliance 
Office of Imperial College London & Imperial College Healthcare NHS Trust. 
 
Indemnity 
Imperial College London holds negligent harm and non-negligent harm insurance policies, 
which apply to the experiments outlined in this protocol. 
 
Sponsor 
Imperial College London will act as the main Sponsor for this study. Delegated responsibilities 
will be assigned to the NHS trusts taking part in this study. 
 

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Funding 
Imperial College London is funding this study through the Imperial College Research 
Fellowship scheme. 
 
 
PUBLICATION POLICY 
 
Results of the study will be disseminated by conference presentation and peer reviewed 
journal publication. This will be in open access formats wherever possible. 

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APPENDIX A 
 
Optical setup and laser safety considerations for bench-top fluorescence 
spectrometer (‘mark I’) 
The optical setup for the bench-top fluorescence spectrometer (‘mark I’) used in the first stages 
of these experiments is shown in Figure 1. The two laser sources have wavelengths of 488 nm 
and 785 nm, which permit excitation of fluorescence from fluorescein/FITC and ICG 
respectively. The output from the two lasers are combined using a dichroic beamsplitter and 
coupled into the excitation channel of the fibre probe. This delivers the excitation light to the 
measurement site and the fluorescence is then collected by the detection channel of the probe 
and routed back to the spectrometer via an emission filter (in order to reject any directly 
scattered excitation light). The fibre probe is electrically insulating to avoid the risk of electric 
shock to the subject or user. All optical components are securely mounted on an optical 
breadboard and are contained within a light-tight box such that the only point at which laser 
radiation is emitted is at the tip of the fibre probe. Neutral density filters are positioned directly 
in front of the two lasers to limit the optical power to a level at which the maximum intensity 
that can be achieved at the distal end of the probe (i.e. when light is optimally coupled into the 
optical fibre) is below the maximum permissible exposure for the skin [31-33]. For both 
excitation wavelengths, this exposure limit will vary depending on the distance of the probe 
from the skin. A range of probe-skin distances will be tested in initial validation experiments to 
determine the optimal distance for spectral permeability measurements, and for each known 
distance the power will be limited accordingly. When the probe is in contact with the skin the 
power will be limited most stringently. In this case, the optical powers at the fibre probe output 
will be restricted to approximately 63 µW at 488 nm and 93 µW at 785 nm. The maximum 
distance from the skin at which the fibre probe will be secured will be approximately 2 mm. At 
this distance the excitation light will illuminate a circle of 600 µm diameter – i.e. it will fully 
illuminate the surface area from which the detection fibres collect light (see Figure 1). In this 
arrangement, as the light is spread over a larger area, the maximum allowable power at the 
distal tip of the probe increases to approximately 565 µW at 488 nm and 836 µW at 785 nm. 
For other fibre-skin separations the maximum permissible power will be calculated in the same 
manner and the laser outputs limited accordingly. Importantly, even with the highest power 
levels discussed above (i.e. at the maximum probe-skin separation), the light that is output 
from the fibre probe will also be below the maximum permissible exposure for the eye when 
the tip of the probe is held at a distance of >10 cm [31-33] (assuming a blink reflex of <0.5 s). 
Thus, in this configuration (with the light-tight box closed), the laser system is eye-safe and 
can be operated without the use of laser safety goggles. In addition, the system has an 
interlock that cuts out the laser emission if the lid is removed. This ensures that the system 
can be safely operated by non-expert users and allows it to be classified as a Class 1 laser 
device. Any re-alignment work is performed by expert laser users only and is carried out in a 
separate locked room with blacked out windows. In this case, the interlock is overridden by the 
expert user and alignment is performed while wearing appropriate laser safety goggles, in 
accordance with local risk assessments. For all clinical measurements, the interlock is 
engaged and the lid is closed such that the laser system is eye-safe, can be classified as a 
Class 1 laser product, and can be operated by non-expert laser users without risk of injury. 
 
 

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APPENDIX B 
 
Discussion of design and safety considerations for the miniaturised (‘mark II’) 
fluorescence spectrometer 
A miniaturised, wearable fluorescence spectrometer will be developed and validated as part 
of this study, with the aim of deploying this ‘mark II’ system to larger scale studies of gut 
permeability. This wearable monitor will use LEDs and PDs for excitation and detection of 
fluorescence. While the exact optical design of the device will be guided by the information 
collected with the ‘mark I’ bench-top system, a likely approach will involve the use of two LEDs 
for excitation of fluorescence (centre wavelengths of approximately 488 nm and 785 nm) and 
four PDs for detection. In this arrangement, optical filters will be placed in front of each of the 
PDs such that they specifically detect light over the following wavelength ranges: 485-495 nm 
(for measurement of the 488 nm excitation power); 505-600 nm (for detection of fluorescein 
fluorescence); 780-790 nm (for measurement of the 785 nm excitation power); and 
800-900 nm (for detection of ICG fluorescence). This would allow detection of fluorescence 
signals from both fluorescein and ICG, as provided by the ‘mark I’ system. Crucially, as the 
‘mark II’ device will be based on LEDs and PDs rather than lasers, optical fibres and a 
commercial spectrometer, it will be straightforward to develop a miniaturised system that can 
be deployed as a wearable sensor. Furthermore, due to the relative simplicity of the system, 
we hope to have manufactured the first ‘mark II’ prototype within a year of beginning 
measurements with the bench-top system (which are nonetheless required in order to guide 
the ‘mark II’ design). 
 
The optical excitation power used with the miniature system will be limited to ensure optical 
safety (i.e. laser/LED safety), and the exact power limits will be determined once the final 
device has been designed/manufactured and the relevant dimensions are known (i.e. the 
illuminated area, the LED-skin distance, etc.). All components will be contained within a 3D-
printed plastic case, which will be sealed to ensure that no electrical components come into 
contact with the skin of the subjects or the researchers. As such, it will be possible for the 
device to be safely operated by non-expert users. The case will also act to secure the device 
such that it is in contact with the patients’ skin at all times during the measurements. The exact 
embodiment will be designed after data has been collected with the bench-top system that 
reveals the most suitable measurement location on the subject’s body. This is likely to entail a 
‘fingerclip’ type device, however skin patches, earlobe clips or other embodiments may also 
be considered, and this will be dependent on the results of measurements made with the ‘mark 
I’ device. Overall, the miniaturised device will be designed such that it is suitable for safe and 
widespread clinical deployment by non-expert users. 
 
 

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REFERENCES 
 
1. 
E. M. M. Quigley, "Leaky gut - concept or clinical entity?," Current Opinion in Gastroenterology, 
32(2), 74-79 (2016) 
2. 
Z. Sun, X. Wang, and R. Andersson, "Role of intestinal permeability in monitoring mucosal 
barrier function. History, methodology, and significance of pathophysiology," Digestive Surgery, 
15(5), 386-397 (1998) 
3. 
M. Camilleri et al., "Intestinal barrier function in health and gastrointestinal disease," 
Neurogastroenterol Motil, 24(6), 503-512 (2012) 
4. 
S. C. Nalle and J. R. Turner, "Intestinal barrier loss as a critical pathogenic link between 
inflammatory bowel disease and graft-versus-host disease," Mucosal Immunology, 8(4), 720-
730 (2015) 
5. 
S. Y. Salim and J. D. Soderholm, "Importance of disrupted intestinal barrier in inflammatory 
bowel diseases," Inflammatory Bowel Diseases, 17(1), 362-381 (2011) 
6. 
S. F. Assimakopoulos et al., "Intestinal barrier dysfunction in hiv infection: Pathophysiology, 
clinical implications and potential therapies," Infection, 42(6), 951-959 (2014) 
7. 
B. Schnabl and D. A. Brenner, "Interactions between the intestinal microbiome and liver 
diseases," Gastroenterology, 146(6), 1513-1524 (2014) 
8. 
E. M. Quigley and H. P. Monsour, "The gut microbiota and nonalcoholic fatty liver disease," 
Seminars in Liver Disease, 35(3), 262-269 (2015) 
9. 
G. Szabo, "Gut-liver axis in alcoholic liver disease," Gastroenterology, 148(1), 30-36 (2015) 
10. 
V. Owino et al., "Environmental enteric dysfunction and growth failure/stunting in global child 
health," Pediatrics, 138(6), (2016) 
11. 
A. J. Thompson et al., "The potential role of optical biopsy in the study and diagnosis of 
environmental enteric dysfunction," Nature Reviews Gastroenterology & Hepatology, 14, 727-
738 (2017) 
12. 
A. Ali, N. T. Iqbal, and K. Sadiq, "Environmental enteropathy," Current Opinion in 
Gastroenterology, 32(1), 12-17 (2016) 
13. 
A. J. Prendergast and P. Kelly, "Interactions between intestinal pathogens, enteropathy and 
malnutrition in developing countries," Current Opinion in Infectious Diseases, 29(3), 229-236 
(2016) 
14. 
D. Mondal et al., "Contribution of enteric infection, altered intestinal barrier function, and 
maternal malnutrition to infant malnutrition in bangladesh," Clin Infect Dis, 54(2), 185-92 (2012) 
15. 
G. T. Keusch et al., "Environmental enteric dysfunction: Pathogenesis, diagnosis, and clinical 
consequences," Clin Infect Dis, 59(Suppl 4), S207-212 (2014) 
16. 
G. T. Keusch et al., "Implications of acquired environmental enteric dysfunction for growth and 
stunting in infants and children living in low- and middle-income countries," Food Nutr Bull, 
34(3), 357-64 (2013) 
17. 
The MAL-ED Network Investigators, "The MAL-ED study: A multinational and multidisciplinary 
approach to understand the relationship between enteric pathogens, malnutrition, gut 
physiology, physical growth, cognitive development, and immune responses in infants and 
children up to 2 years of age in resource-poor environments," Clinical Infectious Diseases, 
59(Suppl 4), S193-S206 (2014) 
18. 
K. G. Dewey and S. Adu-Afarwuah, "Systematic review of the efficacy and effectiveness of 
complementary feeding interventions in developing countries," Matern Child Nutr, 4(Suppl 1), 
24-85 (2008) 
19. 
R. J. Crane, K. D. Jones, and J. A. Berkley, "Environmental enteric dysfunction: An overview," 
Food Nutr Bull, 36(1 Suppl), S76-87 (2015) 
20. 
S. Grantham-McGregor et al., "Developmental potential in the first 5 years for children in 
developing countries," Lancet, 369(9555), 60-70 (2007) 
21. 
A. M. Veitch et al., "Tropical enteropathy: A t-cell-mediated crypt hyperplastic enteropathy," 
European Journal of Gastroenterology & Hepatology, 13(10), 1175-1181 (2001) 
22. 
P. Kelly et al., "Responses of small intestinal architecture and function over time to 
environmental factors in a tropical population," Am J Trop Med Hyg, 70(4), 412-419 (2004) 

GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
 
 
 
 
39 
GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
23. 
W. A. Petri, Jr., C. Naylor, and R. Haque, "Environmental enteropathy and malnutrition: Do we 
know enough to intervene?," BMC Med, 12, 187 (2014) 
24. 
I. Trehan et al., "New insights into environmental enteric dysfunction," Arch Dis Child, 101(8), 
741-744 (2016) 
25. 
D. M. Denno et al., "Use of the lactulose to mannitol ratio to evaluate childhood environmental 
enteric dysfunction: A systematic review," Clin Infect Dis, 59(Suppl 4), S213-219 (2014) 
26. 
L. Wang et al., "Methods to determine intestinal permeability and bacterial translocation during 
liver disease," J Immunol Methods, 421, 44-53 (2015) 
27. 
B. R. Westrom et al., "Intestinal transmission of macromolecules (bsa and fitc-labelled dextrans) 
in the neonatal pig. Influence of age of piglet and molecular weight of markers," Biol Neonate, 
46(1), 20-26 (1984) 
28. 
G. M. Ekstrom et al., "The uptake of fluorescein-conjugated dextran 70,000 by the small 
intestinal epithelium of the young rat and pig in relation to macromolecular transmission into the 
blood," Journal of Developmental Physiology, 10(3), 227-233 (1988) 
29. 
A. J. Thompson et al., "In vivo measurements of diffuse reflectance and time-resolved 
autofluorescence emission spectra of basal cell carcinomas," Journal of Biophotonics, 5(3), 
240-254 (2012) 
30. 
S. Coda et al., "Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and 
diseased colon measured ex vivo using a fiber-optic probe," Biomedical Optics Express, 5(2), 
515-538 (2014) 
31. 
International Commission on Non-Ionizing Radiation Protection (ICNIRP), "Guidelines on limits 
of exposure to laser radiation of wavelengths between 180 nm and 1,000 microns," Health 
Physics, 71(5), 804-819 (1996) 
32. 
International Commission on Non-Ionizing Radiation Protection (ICNIRP), "Revision of 
guidelines on limits of exposure to laser radiation of wavelengths between 400 nm and 1.4 
microns," Health Physics, 79(4), 431-440 (2000) 
33. 
International Commission on Non-Ionizing Radiation Protection (ICNIRP), " ICNIRP guidelines 
on limits of exposure to laser radiation of wavelengths between 180 nm and 1,000 microns," 
Health Physics, 105(3), 271-295 (2013) 
34. 
A. P. Watson and E. S. Rosen, "Oral fluorescein angiography: Reassessment of its relative 
safety and evaluation of optimum conditions with use of capsules," British Journal of 
Ophthalmology, 74(8), 458-461 (1990) 
35. 
Medicines and Healthcare products Regulatory Agency (MHRA), Public assessment report - 
fluorescein sodium 100 mg/ml, solution for injection, in PL 41349/0001, Medicines and 
Healthcare products Regulatory Agency (MHRA), Editor, 2015 
36. 
Medicines and Healthcare products Regulatory Agency (MHRA), Summary of product 
characteristics - fluorescein sodium 100 mg/ml, solution for injection, in PL 43956/0002, 
Medicines and Healthcare products Regulatory Agency (MHRA), Editor, 2015 
37. 
Q. T. Nguyen and R. Y. Tsien, "Fluorescence-guided surgery with live molecular navigation — 
a new cutting edge," Nature Reviews Cancer, 13, 653-662 (2013) 
38. 
M. J. Noble, H. Cheng, and P. M. Jacobs, "Oral fluorescein and cystoid macular oedema: 
Detection in aphakic and pseudophakic eyes," The British Journal of Ophthalmology, 68(4), 
221-224 (1984) 
39. 
Ophthalmic Imaging Association (OIA), Fluorescein and indocyanine dyes for angiography, 
http://www.oia.org.uk/oldsite/pdf/FluIndo.pdf  
40. 
MedCircle, 
Knowledgebase 
- 
fluorescein, 
https://www.medcircle.com/documents/2724-
fluorescein (2017) 
41. 
T. Hara, M. Inami, and T. Hara, "Efficacy and safety of fluorescein angiography with orally 
administered sodium fluorescein," American Journal of Ophthalmology, 126(4), 560-564 (1998) 
42. 
D. Squirrell et al., "Oral fluorescein angiography with the scanning laser ophthalmoscope in 
diabetic retinopathy: A case controlled comparison with intravenous fluorescein angiography," 
Eye, 19, 411-417 (2004) 
43. 
R. V. Azad et al., "Comparative evaluation of oral fluorescein angiography using the confocal 
scanning laser ophthalmoscope and digital fundus camera with intravenous fluorescein 

GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
 
 
 
 
40 
GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
angiography using the digital fundus camera," Clinical & Experimental Ophthalmology, 34(5), 
425-429 (2006) 
44. 
G. Barteselli et al., "Safety and efficacy of oral fluorescein angiography in detecting macular 
edema in comparison with spectral domain optical coherence tomography," Retina, 33(8), 1574-
1583 (2013) 
45. 
J. S. Kelley et al., "Retinal fluorograms using oral fluorescein," Ophthalmology, 87(8), 805-11 
(1980) 
46. 
F. P. Kinsella and D. J. Mooney, "Anaphylaxis following oral fluorescein angiography," American 
Journal of Ophthalmology, 106(6), 745-746 (1988) 
47. 
F. Gómez-Utla, C. Gutiérrez, and I. Seoane, "Severe anaphylactic reaction to orally 
administered fluorescein," American Journal of Ophthalmology, 112(1), 94 (1991) 
48. 
K. Kuroda et al., "Intra-arterial injection fluorescein videoangiography in aneurysm surgery," 
Operative Neurosurgery, 72(Suppl 2), 141-150 (2013) 
49. 
M. B. Wallace et al., "The safety of intravenous fluorescein for confocal laser endomicroscopy 
in the gastrointestinal tract," Alimentary Pharmacology & Therapeutics, 31(5), 548-552 (2010) 
50. 
Medicines and Healthcare products Regulatory Agency (MHRA) Summary of product 
characteristics - verdye (icg) 5 mg/ml injection, in PL 44791/0001, Medicines and Healthcare 
products Regulatory Agency (MHRA), Editor, 2016 
51. 
A. L. Vahrmeijer et al., "Image-guided cancer surgery using near-infrared fluorescence," Nature 
Reviews Clinical Oncology, 10, 507-518 (2013) 
52. 
J. T. Alander et al., "A review of indocyanine green fluorescent imaging in surgery," International 
Journal of Biomedical Imaging, 2012, 26 (2012) 
53. 
MedCircle, Knowledgebase - indocyanine green, https://www.medcircle.com/documents/2848-
indocyanine-green (2017) 
54. 
S. Sou et al., "Differentiating enterocutaneous fistulae from suture abscesses complicating 
crohn’s disease using oral administration of indocyanine green," Journal of Gastroenterology 
and Hepatology, 21(12), 1850-1853 (2006) 
55. 
G. Paumgartner, "The handling of indocyanine green by the liver," Schweiz Med Wochenschr, 
105(17 Suppl), 1-30 (1975) 
56. 
E. G. Atkinson et al., "Molecular size of retinal vascular leakage determined by fitc-dextran 
angiography in patients with posterior uveitis," Eye, 5, 440-446 (1991) 
57. 
P. G. Wheeler, I. S. Menzies, and B. Creamer, "Effect of hyperosmolar stimuli and coeliac 
disease on the permeability of the human gastrointestinal tract," Clinical Science and Molecular 
Medicine, 54(5), 495-501 (1978) 
58. 
U. Schroder, K. E. Arfors, and O. Tangen, "Stability of fluorescein labeled dextrans in vivo and 
in vitro," Microvascular Research, 11(1), 33-39 (1976) 
59. 
B. Åberg, "Breakdown of dextran by human feces," Scandinavian Journal of Clinical and 
Laboratory Investigation, 5(1), 37-38 (1953) 
60. 
Medicines and Healthcare products Regulatory Agency (MHRA), Summary of product 
characteristics - rescueflow, solution for injection (dextran 70), in PL 41284/0001, Medicines 
and Healthcare products Regulatory Agency (MHRA), Editor, 2012 
61. 
C. E. Zinderman, L. Landow, and R. P. Wise, "Anaphylactoid reactions to dextran 40 and 70: 
Reports to the united states food and drug administration, 1969 to 2004," J Vasc Surg, 43(5), 
1004-9 (2006) 
62. 
K. G. Ljungstrom et al., "Adverse reactions to dextran in sweden 1970-1979," Acta Chir Scand, 
149(3), 253-262 (1983) 
63. 
J.-R. Laporte et al., "An epidemiologic study of severe anaphylactic and anaphylactoid reactions 
among hospital patients: Methods and overall risks. The international collaborative study of 
severe anaphylaxis," Epidemiology, 9(2), 141-6 (1998) 
64. 
S. Miura, Y. Teramura, and H. Iwata, "Encapsulation of islets with ultra-thin polyion complex 
membrane through poly(ethylene glycol)-phospholipids anchored to cell membrane," 
Biomaterials, 27(34), 5828-5835 (2006) 
65. 
W. Lu et al., "Targeted photothermal ablation of murine melanomas with melanocyte-stimulating 
hormone analog-conjugated hollow gold nanospheres," Clin Cancer Res, 15(3), 876-886 (2009) 

GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
 
 
 
 
41 
GutPerm – Protocol Version 4.2A: 01-May-2019 
IRAS ID: 242462 
 
66. 
M. Vila et al., "Cell uptake survey of pegylated nanographene oxide," Nanotechnology, 23(46), 
465103 (2012) 
67. 
J. Linares et al., "Endocytic mechanisms of graphene oxide nanosheets in osteoblasts, 
hepatocytes and macrophages," ACS Applied Materials & Interfaces, 6(16), 13697-706 (2014) 
68. 
X. Chen et al., "Subcellular fate of a fluorescent cholesterol-poly(ethylene glycol) conjugate: An 
excellent plasma membrane imaging reagent," Langmuir, 32(39), 10126-10135 (2016) 
69. 
R. Ohgaki et al., "Ratiometric fluorescence imaging of cell surface ph by poly(ethylene glycol)-
phospholipid conjugated with fluorescein isothiocyanate," Scientific Reports, 7(1), 17484 (2017) 
70. 
D. Cui et al., "Gastrin-releasing peptide receptor-targeted gadolinium oxide-based 
multifunctional nanoparticles for dual magnetic resonance/fluorescent molecular imaging of 
prostate cancer," International Journal of Nanomedicine, 12, 6787-6797 (2017) 
71. 
A. D'Souza A and R. Shegokar, "Polyethylene glycol (PEG): A versatile polymer for 
pharmaceutical applications," Expert Opinion on Drug Delivery, 13(9), 1257-1275 (2016) 
72. 
Biolab Medical Unit - Nutritional and Environmental Medicine, Gut permeability profile, 
http://www.biolab.co.uk/print.php/cmsid__biolab_test/Gut_Permeability_profile (2013) 
73. 
Biolab Medical Unit - Nutritional and Environmental Medicine, Investigation of gut permeability, 
http://www.biolab.co.uk/docs/peg.pdf (2013) 
74. 
MedCircle, 
Knowledgebase 
- 
polyethylene 
glycol 
3350, 
https://www.medcircle.com/documents/3560-polyethylene-glycol-3350 (2018) 
75. 
MedCircle, 
Knowledgebase 
- 
polyethylene 
glycol 
- 
electrolyte 
solution, 
https://www.medcircle.com/documents/3200-polyethylene-glycol-electrolyte-solution (2018) 
76. 
Medicines and Healthcare products Regulatory Agency (MHRA) Public assessment report - 
dulcosoft 10 g powder for oral solution in sachet; macrogol 4000 10 g powder for oral solution 
in sachet (macrogol 4000), in PL 23022/0112-0113, Medicines and Healthcare products 
Regulatory Agency (MHRA), Editor, 2015 
77. 
H. B. Co-Minh et al., "Anaphylactic shock after oral intake and contact urticaria due to 
polyethylene glycols," Allergy, 62(1), 92-93 (2007) 
78. 
S. H. Lee et al., "Anaphylactic shock caused by ingestion of polyethylene glycol," Intestinal 
Research, 13(1), 90-94 (2015) 
79. 
S.-H. Lee et al., "Anaphylaxis to polyethylene glycol (colyte®) in a patient with diverticulitis," 
Journal of Korean Medical Science, 31(10), 1662-1663 (2016) 
80. 
K. Wylon, S. Dölle, and M. Worm, "Polyethylene glycol as a cause of anaphylaxis," Allergy, 
Asthma & Clinical Immunology, 12(67), (2016) 
81. 
M. Willems, A. O. Quartero, and M. E. Numans, "How useful is paracetamol absorption as a 
marker of gastric emptying? A systematic literature study," Digestive Diseases and Sciences, 
46(10), 2256-2262 (2001) 
82. 
M. F. Laker and I. S. Menzies, "Increase in human intestinal permeability following ingestion of 
hypertonic solutions," Journal of Physiology, 265(3), 881-894 (1977) 
83. 
F. Andre et al., "Assessment of the lactulose-mannitol test in crohn's disease," Gut, 29(4), 511-
515 (1988) 
84. 
S. D. Johnston et al., "Lactulose-mannitol intestinal permeability test: A useful screening test for 
adult coeliac disease," Annals of Clinical Biochemistry, 37(Pt 4), 512-519 (2000)