Insufficient Cellular Oxygen in ICU Patients With Anaemia: the INOX ICU-2 Study
Conditions: Critical Care, Anemia, Erythrocyte Transfusion, Oxygen, Mitochondria
Sponsor: Sanquin-LUMC J.J van Rood Center for Clinical Transfusion Research

NL59512.058.16 / P16.303 
 
INOX ICU-2 study 
 
 
 
Last updated on: 09-12-2019 
 
Summary of changes made to the 
original protocol listed  
 
Original protocol: 
Research protocol – v2.4 31-03-2017 
 
Changes leading to protocol v2.5 28-06-2017: 
- 
Not having a central venous catheter in situ has been dismissed as an exclusion criterium for 
the pilot study, since it will not affect the feasibility of the mitochondrial oxygenation 
measurement. 
 
Changes leading to protocol v3.0 23-01-2018: 
- 
Study group information updated and another member of the study group added to the 
protocol. 
- 
Correction of spelling and grammatical errors. 
- 
Addition of insufficient comprehensibility of the Dutch language as an exclusion criterium, 
since informed consent procedure is in Dutch. 
- 
Changes made to the study design due to logistical problems encountered during the pilot 
study 
o We saw that inclusions were missed in patients receiving red blood cell transfusion 
in the first hours after cardiothoracic surgery, due to not being able to asked 
informed consent to the still sedated patient. Therefore, informed consent to 
cardiothoracic patients, which will be admitted to the ICU post-surgery, is asked 1 
day before the surgery takes place.  
o Due to heterogeneity in red blood cell transfusion time, it has now been 
protocolized that red blood cell transfusions will be given in one hour. The clinician 
can still deviated from this time frame is the condition of the patient does not 
permit a one hour lead time. 
o During the pilot study, the investigators noticed missing information regarding 
smoking habit and New York Heart Association in almost all patients due to no 
standardized recording of these data. 
o There were 8 measurement moments during the pilot study in the 24 hours 
following red blood cell transfusion. Due to logistics and stability of the 
measurement during certain time points, the measurement moments were reduced 
to 6 measurement moments. The dismissed time points were 15 minutes after the 
end of red blood cell transfusion and 120 minutes after the end of red blood cell 
transfusion. 
o No IN vivo optical spectroscopy of cerebral oxygenation device can be used during 
the main study due to logistical problems. This measurement has been deleted from 
the protocol. 
o Sample size calculation is updated with the results of the pilot study. 
o The monitor of the study is specified and the monitorplan is updated. 
 
Changes leading to protocol v4.0 19-10-2018: 
- 
Not having a central venous catheter has been dismissed as an exclusion criterium in the 
main study as well due to low inclusion rate. 

NL59512.058.16 / P16.303 
 
INOX ICU-2 study 
 
 
 
Last updated on: 09-12-2019 
 
- 
Due to the slow inclusion rate the informed consent procedure has been updated. Now, all 
ICU patients admitted to the ICU and expected to stay at least 24 hours in the ICU are asked 
for participation in the study. If the patient is not able to give her/his consent, the legal 
representative will be asked. 
 
Changes leading to protocol v5.0 27-02-2019: 
- 
Due to the variable time frame between admission to the ICU and being able to do informed 
consent with the patient and/or their legal representative, possible research subjects were 
missed. Therefore, the informed consent procedure was optimized. If the patient or legal 
representative could not have been reached in time for written informed consent, while the 
patient met the eligibility criteria, the patient was included with deferred consent 
procedure. Informed consent was sought within 48 hours after inclusion of the patient. 
- 
The protocol has been updated to the latest regulations regarding privacy (General Data 
Protection Regulation).  

INsufficient cellular OXygen in ICU 
patients with anaemia: 
the INOX ICU–2 study 
Coordinating investigator 
Msc. M. Baysan, Department of Intensive Care, Clinical Epidemiology and Transfusion Research, 
Leiden University Medical Center and Sanquin Research 
Principal Investigators 
▪
Dr. M.S. Arbous, Department of Intensive Care and Department of Clinical Epidemiology,
Leiden University Medical Center
▪
Prof. Dr. J.G. van der Bom, Department of Clinical Epidemiology and Clinical Transfusion
Research, Sanquin Research
▪
Prof. Dr. N.P. Juffermans, Department of Intensive Care, Amsterdam Medical Center
▪
Dr. E.G. Mik, Department of Anaesthesiology, Erasmus University Medical Center
Independent Physician 
Drs. J. van Paassen, Department of Intensive Care, Leiden University Medical Center 
Institute: Departments of Intensive Care, Clinical Epidemiology, and Immunohematology and Blood 
Transfusion of the Leiden University Medical Centre, Leiden, The Netherlands 
Sponsor: Leiden University Medical Center 
Funding: Sanquin Research (PPOC 16-31) 
ABR number:  NL59512.058.16 
Protocol number: P16.303 
Version: 5.0 
Date: 27-02-2019 
Trial registration number: NCT03092297
Contact details 
M Baysan, MD 
Department of Intensive Care 
Location J-04-027, LUMC 
Albinusdreef 2 
2333 ZA Leiden 
Telephone: +31715297274 
Email: M.Baysan@lumc.nl  

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TITLE:  Insufficient cellular oxygen in ICU patients with anaemia: the INOX ICU-2 Study 
Protocol ID 
INOX ICU-2 Study 
Short title 
Insufficient cellular oxygen in ICU patients with anaemia: 
the INOX ICU-2 Study 
Version 
5.0 
Date 
27-02-2019 
Coordinating investigator 
Msc. M. Baysan   
Leiden University Medical Center and Sanquin Research 
Leiden 
Department of Intensive Care, Clinical Epidemiology, and 
Clinical Transfusion Research 
Plesmanlaan 1A, 2333 BZ Leiden, the Netherlands 
(Sanquin) 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands (LUMC) 
Room: J-04-027 
Telephone: +31715297274 
Email: M.Baysan@lumc.nl  
 
Principal investigators 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prof. Dr. J.G. van der Bom 
Leiden University Medical Center and Sanquin Research 
Leiden 
Department of Clinical Epidemiology and Clinical 
Transfusion Research 
Plesmanlaan 1A, 2333 BZ Leiden, the Netherlands 
(Sanquin) 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands (LUMC) 
Room C-07-093(LUMC)  
Telephone: +31715268871 
Email: J.G.van_der_Bom@lumc.nl   
 
Dr. M.S. Arbous 
Leiden University Medical Center 
Department of Intensive Care and Department of Clinical 
Epidemiology 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands 
Room: J-04-026 
Telephone: +31715261678 
Email: M.S.Arbous@lumc.nl  
 

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Multicenter research 
Principal investigator per site: 
Prof. Dr. N.P. Juffermans 
Amsterdam Medical Center 
Department of Intensive Care 
Meibergdreef 9, 1105 AZ Amsterdam-Zuidoost, the 
Netherlands 
Email: n.p.juffermans@amc.uva.nl  
 
Dr. E.G. Mik 
Erasmus University Medical Center 
Department of Anesthesiology 
's-Gravendijkwal 230, 3015 CE Rotterdam, the 
Netherlands 
Email: e.mik@erasmusmc.nl  
Other members research group 
Dr. T.J. van Brakel 
Leiden University Medical Center 
Department of cardiothoracic surgery 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands 
Room: D-06-031 
Telephone: +31715297790 
Email: T.J.van_Brakel@lumc.nl  
 
Sponsor  
Leiden University Medical Center 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands 
Subsidising party 
Sanquin Research 
Plesmanlaan 1A, 2333 BZ Leiden, the Netherlands 
Independent expert  
Drs. J. van Paassen  
Department intensive care 
Albinusdreef 2, 2333 ZA Leiden, the Netherlands 
Room: J-04-024  
Telephone: +31715262782 
Email: J.van_Paassen@lumc.nl 
 
 
 
 
 

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TABLE OF CONTENTS 
PROTOCOL SIGNATURE SHEET ......................................................................................................... 5 
SUMMARY .............................................................................................................................................. 6 
2. OBJECTIVES ...................................................................................................................................... 9 
2.1 GENERAL ......................................................................................................................................... 9 
2.2 ENDPOINTS ...................................................................................................................................... 9 
3. DESIGN AND POPULATION ........................................................................................................... 11 
3.1 STUDY DESIGN ............................................................................................................................... 11 
3.2. STUDY POPULATION ...................................................................................................................... 11 
4. 
TREATMENT OF SUBJECTS ...................................................................................................... 12 
INVESTIGATIONAL PRODUCT: PPIX-TSLT WITH COMET .......................................................................... 12 
USE OF CO-INTERVENTION .................................................................................................................... 13 
ESCAPE MEDICATION ............................................................................................................................ 14 
5. 
METHODS ..................................................................................................................................... 15 
5.1 STUDY PROCEDURES ..................................................................................................................... 15 
5.1.1 General ................................................................................................................................. 15 
5.1.2 Clinical Care ......................................................................................................................... 16 
5.2 WITHDRAWAL OF INDIVIDUAL SUBJECTS ........................................................................................... 16 
5.3 Data collection ......................................................................................................................... 16 
5.4 STUDY PARAMETERS ...................................................................................................................... 19 
6. STATISTICAL CONSIDERATION .................................................................................................... 20 
6.1 POWER ESTIMATE .......................................................................................................................... 20 
6.2 STATISTICAL METHODS TO BE EMPLOYED ........................................................................................ 20 
7. SAFETY REPORTING ...................................................................................................................... 21 
7.3 Adverse events (AEs) .............................................................................................................. 21 
7.4 Serious adverse events (SAEs)............................................................................................... 21 
7.6 ANNUAL SAFETY REPORT ................................................................................................................ 23 
7.7 FOLLOW-UP OF ADVERSE EVENTS ................................................................................................... 23 
8. ETHICAL CONSIDERATIONS ......................................................................................................... 25 
8.1 REGULATION STATEMENT ............................................................................................................... 25 
8.2 RECRUITMENT AND CONSENT ......................................................................................................... 25 
8.3 OBJECTION BY MINORS OR INCAPACITATED SUBJECTS ...................................................................... 26 
8.4 BENEFITS AND RISK ASSESSMENT, GROUP RELATEDNESS ................................................................. 26 
8.5 COMPENSATION FOR INJURY ........................................................................................................... 26 
9. ADMINISTRATIVE ASPECTS AND PUBLICATION ....................................................................... 27 
9.1 HANDLING AND STORAGE OF DATA AND DOCUMENTS ........................................................................ 27 
9.3 AMENDMENTS ................................................................................................................................ 27 
9.4 ANNUAL PROGRESS REPORT ........................................................................................................... 27 
9.5 END OF STUDY REPORT .................................................................................................................. 28 
9.6 PUBLIC DISCLOSURE AND PUBLICATION POLICY ................................................................................ 28 
10. STRUCTURED RISK ANALYSIS ................................................................................................... 29 
11. LIST OF ABBREVIATIONS ............................................................................................................ 32 
12. REFERENCES ................................................................................................................................ 33 
13. APPENDIX 1: BACKGROUND OF THE MEASUREMENTS ........................................................ 36 
14. APPENDIX 2: OVERVIEW OF MEASUREMENTS ........................................................................ 40 
15.APPENDIX 3: SKIN PAD ........................................................................................................ 433 
16.APPENDIX 4: MONITORPLAN ACCORDING TO NFU GUIDELINES ........................ 435 
 
 
 
 

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Summary 
 
Rationale: Evidence is increasing that in some cases a haemoglobin trigger of 7-8g/dl may be too low 
and that an individualized red cell transfusion strategy may benefit critically ill patients. New studies 
have shown the potential of a protoporphyrin IX-triple state lifetime technique to measure 
mitochondrial oxygenation tension (mitoPO2) in vivo, which possibly is an early indicator of oxygen 
disbalance in the cell and therefore a physiological trigger for red cell transfusion. 
Objectives: 1. To determine the feasibility of using mitoPO2 and the variability of mitoPO2 
measurements in critically ill intensive care unit (ICU) patients who are about to receive a red cell 
transfusion. 2. To describe the effects of red cell transfusion and the associated change in 
haemoglobin on mitoPO2 and on other physiologic measures of tissue oxygenation and oxygen 
balance 3.To describe the association between mitoPO2 and vital organ functions and clinical 
outcomes. 
Study design: pilot study followed by a prospective multicentre cohort study 
Study population: ICU patients with anaemia in whom a red cell transfusion is planned. 
Main study endpoints: 
Primary endpoint: 
- 
Variability of mitoPO2 before and after red cell transfusion. This will be compared to 
traditional parameters used to measure tissue oxygenation and oxygen balance 
Secondary endpoints: 
- 
Association of mitoPO2 with (ischemic) organ damage 
- 
Association of mitoPO2 with the microcirculation 
- 
Duration of mechanical ventilation 
- 
Need for  continuous renal replacement therapy (CRRT) 
- 
Length of ICU-stay 
- 
Length of hospital-stay 
- 
ICU-mortality  
- 
90-day mortality 
 
Study procedure: Included patients will undergo red cell transfusion as planned. However, red cell 
transfusion will be delayed for 2 hours. At multiple predefined moments, before and after red cell 
transfusion, data collection including blood samples and measurements of mitoPO2 will take place. It 
will be directly processed. 
Nature and extent of the burden and risks associated with participation, benefit and group 
relatedness: The risks are moderate in this study with no SAE known. The burden for the ICU-
patients is moderate since it’s a non-invasive measurement. The normal clinical practice will continue 
and will not be altered. Since most ICU-patients have in this very controlled environment a central 
venous catheter and arterial catheter in situ, they are the most ideal patients to investigate mitoPO2 
feasibility, accuracy and associations with other physiological parameters.

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1. Introduction and rationale 
 
Depending on the duration of critical illness eventually all critically ill patients develop anaemia which 
may severely affect their recuperation.1 Deleterious effects of severe anaemia include a generalized 
decrease in oxygen carrying capacity and potentially ensuing multi-organ failure. Whereas in the old 
days clinicians tended to transfuse red cells as soon as haemoglobin(Hb) levels were slightly below 
normal, this has radically changed since 1999. In 1999 the landmark TRICC trial showed that critically 
ill patients that received red cell transfusions at a Hb of 9 g/dl were not better off than patients that 
received red cell transfusions only after their Hb reached 7 g/dl.2 The notion that critically ill patients 
with Hb above 7 or 8 g/dl, might not profit from red cell transfusions has been confirmed by other 
studies and is now widely adopted in clinical guidelines.3-5 Yet, there is also increasing evidence that 
in some cases a Hb transfusion trigger of 7- 8 g/dl may be too low. Patients with an ischemic brain, 
spinal cord, or myocardium, with renal insufficiency, or older patients might thus profit from red cell 
transfusion at higher Hb levels.6,7 Whether these findings are solid enough to change clinical practice 
is heavily debated.8,9 These uncertainties about efficacy and safety of transfusion lead to undesirable 
ambiguities in ICU transfusion practice.  
 
Mik et al. has recently introduced the protoporphyrin IX-triplet state lifetime technique as the first 
method to measure mitochondrial oxygen tension (mitoPO2) in living cells and tissues.10 Experimental 
results have shown mitoPO2’s robustness and clinical potential. 10-13 Animal research has shown that: 
(i) cutaneous mitoPO2 decreases abruptly during haemodilution;(ii) abrupt decrease in mitoPO2 
precedes lactate increase (as a consequence of a lack of tissue oxygenation) and hemodynamic 
instability; (iii) abrupt decrease in mitoPO2 occurs at different (low) Hb for different individuals; (iv) 
cutaneous mitoPO2 could be a marker for the physiological limits of isovolemic haemodilution; (v) 
cutaneous mitoPO2 could be an indicator for the need of a red cell transfusion; (vi) if haemodilution is 
stopped after a predefined drop in mitoPO2, mitoPO2 remains stable; (vii) but after 2 hours kidney 
failure occurs and lactate rises; (viii) if instead a red cell transfusion is administered after this drop, 
mitoPO2 rises again and levels of lactate and organ functions remain normal.13-15 Thus, mitoPO2 was 
able to predict the need for a red cell transfusion. 
 
In the ongoing INOX ICU-1 study, a decision tool for red blood cell transfusion is being developed in 
critically ill patients using retrospective ICU data. The proposed INOX ICU-2 project is a phase 1 
diagnostic study examining whether mitoPO2, a recently developed test measuring mitochondrial 
oxygen, is a potentially useful candidate to improve the INOX ICU prediction tool. The results of the 
INOX ICU-2 will reveal whether mitoPO2 could be used to further tailor red cell transfusion strategies 
to the individual patient and ultimately reduce the harmful effects of both under- and over-transfusion 
in ICU patients.  
 
This is the very first research on mitoPO2’s clinical applicability in critically ill patients with anaemia 
and the first attempt to personalize red cell transfusion decisions in the ICU based on individual 

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assessment of the oxygen balance on a mitochondrial level, i.e. the ability of oxygen delivery to 
tissues to fulfil actual oxygen demand. We hypothesize that mitoPO2 can also be used as an early 
predictor of organ dysfunction. If this is true, mitoPO2 might be used to administer or postpone red cell 
transfusion in patients with anaemia. 
 
The results of the proposed project cannot be immediately translated to clinical practice. Using these 
results, we will design a phase 2 diagnostic study, most probably a randomized clinical trial that will 
yield applied knowledge with respect to personalizing red cell transfusion. Application will be in ICU 
patients with anaemia who might or might not profit from red cell transfusions. It will lead to a reduction 
of both over- and under- transfusion. 
 
 
 

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2. Objectives 
 
2.1 General 
The ultimate aim of the INOX ICU project is to tailor red cell transfusion strategies to individual ICU 
patients with anaemia with respect to the potential risks and benefits. After the proposed project we 
will know the essential test characteristics needed to ultimately assess the added value of the 
mitoPO2 for the transfusion decision. With these results it will be possible to proceed to the next 
phase. In the next phase (not part of this project) we will compare whether clinical outcomes of 
patients in whom transfusion strategies are determined using mitoPO2 results are better than clinical 
outcomes of patients in whom transfusion strategies are determined without mitoPO2. 
 
2.2 Endpoints 
In this phase 1 diagnostic study we aim to investigate mitoPO2 test’s feasibility, accuracy, and 
associations with other physiological parameters of oxygen balance and tissue oxygenation and with 
ischemic organ damage in critically ill patients with anaemia. 
 
Specific objectives: 
1. To determine the feasibility and variability of mitoPO2 measurement in critically ill ICU patients 
before and after a red cell transfusion. 
2. To describe the effects of red cell transfusion and the associated change in Hb on mitoPO2 and on 
other physiologic measures of tissue oxygenation and oxygen balance such as, arterial oxygen 
saturation, central venous oxygen saturation (ScvO2, measured in either the jugular or subclavian 
vein, if applicable), lactate, and DO2 minus VO2 (difference between oxygen consumption and 
delivery). 
3. To describe the association between mitoPO2 and vital organ functions, as assessed by SOFA 
(Sepsis related Organ Failure Assessment) score, duration of mechanical ventilation, duration of 
vasopressor therapy, myocardial ischemia, serum creatinine, RIFLE classification for renal function, 
need for renal replacement therapy, Glasgow coma score, RASS, ICDSC score. 
4. To describe the association between mitoPO2 measurements and the microcirculation 
 
With this in mind, we will assess the following parameters: 
 
1. Prognostic: duration of mechanical ventilation, need for renal replacement therapy, ICU-stay, 
hospital stay, ICU-mortality, 90-day mortality, measurements of ischemic damage (particular 
in patients following transfusion).  
2. Etiologic: levels and time course of markers of ischemia-reperfusion in relation to the clinical 
course (such as duration of mechanical ventilation, need for renal replacement therapy, ICU-
stay, hospital-stay, ICU-mortality, 90-day mortality, measurements of ischemic damage) 
particular in patients following transfusion. 

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3. Methodologic: the value of mitoPO2 in patients receiving red cell transfusion compared to 
markers of hypoperfusion and oxygen balance used in standard critical care: lactate, ScvO2, 
arterial saturation (SaO2), arterial oxygen tension (PaO2), venous oxygen tension (PvO2), 
cardiac index (CI)). 
 
 

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3. Design and population 
 
3.1 Study design  
First, we performed a pilot study in the LUMC among 20 ICU patients with anaemia in whom a red cell 
transfusion was administered. After assessment of the feasibility of the mitoPO2 measurement and 
sample size, we will now continue with a prospective cohort study. This study will be performed in the 
ICUs of the Leiden University Medical Center (dr. M.S. Arbous), the Amsterdam Medical Center (Prof. 
dr N.P. Juffermans and the Erasmus University Medical Center (dr E.G. Mik) on patients with anaemia 
in whom an arterial catheter is already in place. MitoPO2 and other parameters will be measured at 
inclusion, before red cell transfusion, and at various time points after red cell transfusion. Appendix II 
presents study design, measurements and outcomes for the pilot study, as well as the multicentre 
prospective cohort study. During the study nurses and clinicians will be blinded for the results of the 
mitoPO2, since the investigator executes the mitoPO2 measurement and will not reveal the results. 
 
3.2. Study population 
Inclusion criteria: All adult patients admitted to the ICU with a Hb below 6.3 mmol/l, who were planned 
to undergo a transfusion of red blood cells were included in the pilot study. In the prospective 
multicentre cohort study, only patients with a Hb below 6.3 mmol/l in whom an arterial catheter is in 
place and in whom a red cell transfusion is planned will be included.  
 
Exclusion criteria:  
-patients without a legal representative to ask for  informed consent  
-patients less than 18 years old  
-pregnant or breast feeding women since there is no adequate data from the use of ALA in pregnant 
or breast feeding women16  
-patients in need of emergency red cell transfusion e.g. bleeding patients 
-patients with porphyria and/or known photodermatosis 
-patients with hypersensitivity to the active substance or to the plaster material of ALA 
-patients with an expected ICU stay <24 hours 
-insufficient comprehensibility of the Dutch language 
 
 
 

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4. Treatment of subjects 
 
Investigational product: PpIX-TSLT with Comet 
The COMET's non-invasive cutaneous mitoPO2 measurements rely on the PpIX-TSLT(protoporphyrin 
IX-triple state lifetime technique). The PpIX-TSLT technique measures oxygen by oxygen-dependent 
quenching of delayed fluorescence lifetime of ALA (5-aminovulinic acid)-induced mitochondrial PpIX. 
PpIX is the final precursor of heme in the heme biosynthesis pathway and is synthetized in the 
mitochondria. The conversion of PpIX to haeme is the rate-limiting step and therefore causes a weak 
delayed fluorescence lifetime. Administration of exogenous ALA enhances PpIX to detectable levels 
and enhances mitochondrial origin of the delayed fluorescence signal.10 Measurements are based on 
the detection of time of extinction of red light emitted by the tissue following excitation with green light 
(figure 1). This technique was first published in 2006.17 The technique has been tested and calibrated 
for use in isolated organs and in vivo.18-20 The PpIX-TSLT technique is described in detail by Mik 
(2013).10  
 
Figure 1: Detected delayed fluorescence. A short lifetime corresponds with high intra cellular oxygen 
concentration and low PO2 values correspond with a long lifetime (Mik, 2013). The lifetime can be translated to 
an oxygen tension with the help of a calibration 
 
The COMET measurement system is an oxygen availability measurement tool that measures oxygen 
tension at the mitochondrial level by means of delayed fluorescence of protoporphyrin IX. The COMET 
monitor is used observationally. It will not be used to treat patients, nor is the information provided by 
the COMET used to alter patient treatment. The COMET measurement system is a non-invasive 
measuring system to determine cellular oxygen availability locally in human skin cells with a high 
concentration of protoporphyrin IX. It is CE marked as a medical device (DEKRA certificate number 
2183975CE01, initially issued on 18 April 2016).  
 
In this study, the mitoPO2 of the skin will be measured. Reliable measurements can only take place 
with sufficient concentrations of protoporphyrin IX in the mitochondria. Therefore, a self-adhesive 
patch containing 8mg of ALA (Alacare®; Spirig AG, Egerkingen, Switzerland) will be placed on the 
anterior chest wall for induction of PpIX, for at least 4 hours before the first measurement can take 
place. To enhance ALA penetration, the skin will be rubbed with alcohol to remove the exposed layers 
of stratum corneum. Hair, if present, will be shaved.21 Following at least 4 hours induction of PpIX, the 
ALA patch will be removed after the outline of the ALA patch is marked. To create steady and reliable 

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measurements, a specially designed plaster will be used to attach the COMET Skin Sensor (COMET 
Skin Sensor, Photonics Healthcare, the Netherland) to the skin. This plaster is referred to as the Skin 
Pad, it is not CE marked, and is adhesive on both sides. One side of the plaster is a silicon skin 
friendly side and the other a strong acrylic adhesive to which the Skin Sensor is attached. 
 
Figure 2: Skin Pad 
 
After placing the skin sensor, calibration measurements will be made to ensure the quality of the 
measurements. Thereto, 5 measurements will be made in the first minute to establish a reliable and 
stable measurement. Occlusion of the microcirculation will be accomplished via local pressure with the 
measurement probe, which we use to validate the actual mitoPO2 measurement. After these 
measurements, mitoPO2 will be a measured each minute for 5 minutes to obtain a mean mitoPO2. To 
ensure that no other variables influence the measurements, measurement characteristics will be 
logged. In this log, the circumstances in which each measurement is made will be documented. 
Known intervening variables include, for example, skin temperature, thus skin temperature will be 
recorded. Other variables recorded in the log will be vasopressor use, dosage of the vasopressor, and 
nurse actions. Following mitoPO2 measurement, the ALA patch will be re-used to protect the exposed 
skin for phototoxicity until the next measurement. After completion of the mitoPO2 measurements the 
exposed skin will be protected from sunlight for 24 hours. An overview of the mitoPO2 measurement is 
given below. 
 
The skin sensor and other parts of the COMET will be cleaned according to cleaning guidelines for 
medical devices in the intensive care unit. In this case, the COMET will be cleaned with 70% ethanol. 
In the case of visible dirt, the COMET will be cleaned with water and soap prior to ethanol 70% 
application. 
Use of co-intervention  
Due to the risk of phototoxicity, it is important for the skin exposed to the ALA patch to be protected 
from sunlight during, between, and for 24 hours following measurements. 
ALA patch (for 
>4 hours)
obtaining a 
stable mitoPO2
mitoPO2 
validation
mean of 
mitoPO2 
measurement 
(log each 
measurement)
protection skin 
with ALA patch 
untill next 
measurements

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Escape medication  
Should adverse effects take place, the skin would need to be protected from further damage by light 
exposure. Possible adverse effects include burning sensation of the skin, pruritis, hyperpigmentation, 
application site erythema, and local pain.22,23 Pain killers could be administered, if local pain would 
persist despite these precautions.  
 

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5. Methods 
5.1 Study procedures 
5.1.1 General 
Since research has shown that approximately 10% of critically ill patients can make autonomous 
decision regarding research participation, most decisions will be made by their substitute decision 
makers, who are already overwhelmed by the medical information of the patient itself. Furthermore, 
studies have shown that obtaining contact with their substitute decision makers can be difficult in 7%, 
no decision maker exist in 6% of the cases. In addition, approximately half of the opportunities to 
recruit patients are missed because of workload and narrow time windows for inclusion. Difficulties in 
recruitment of eligible patient can lead to delayed identification of (in)effective treatment/diagnostics 
and limitation of generalizability. Alternative consent models are therefore needed to improve 
recruitment and minimize withdrawal.24,25 Therefore, we will first ask all ICU patients admitted to the 
ICU, or their legal representatives, expected to stay at the ICU for longer than 24 hours for 
participation in our study. Patients admitted to the cardiothoracic department which will be admitted to 
the ICU post-operatively, will be asked for an informed consent on the day before the cardiothoracic 
operation. An information leaflet will be given and informed consent will be sought. Patients or their 
legal representatives will be told that a person can only participate in the study if he/she meets the 
inclusion criteria. However, if no legal representative could have been reached in person for written 
informed consent, while the patient meet the eligibility criteria, the patient will be included in the study 
and deferred informed consent will be sought within 48 hours after oral consent has been given. If 
written informed consent is available or oral consent given, at the moment the decision is made to 
administer a red cell transfusion, the self-adhesive ALA-patch will be placed. Due to logistics, the 
normal time elapsing between the decision of a non-emergency red cell transfusion and the actual 
administration is 2 to 3 hours. The induction time of PpIX is at least 4 hours, therefore red cell 
transfusion will be delayed by 1 to 2 hours. At no time red cell transfusion will be delayed in order to 
have enough time to induce PpIX if the patient is clinically in need of a red cell transfusion. To ensure 
this, the clinician responsible for the patient, and not the researcher, has the final decision in the timing 
of the red cell transfusion. 
 
After inclusion of a patient meeting the eligibility criteria, each patient will be assigned an unique study 
number.  
 
To determine the reliability of the mitoPO2 measurement in critically ill ICU patients with anaemia who 
will receive transfusion, we first assessed the variance of mitoPO2 from repeated mitoPO2 values 
before and after transfusion in 20 ICU patients. Next, using this variance, we estimated the number of 
patients needed to perform a reliable multicentre study26, which we subsequently will perform.  
 
To examine the effects of red cell transfusion and the associated change in Hb on mitoPO2, and on 
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and lactate, we will compare values of all parameters before with the values after red cell transfusion. 
In addition we will perform similar analyses in subgroups of Hb and reason of admission to assess 
whether the observed effect of red cell transfusion on the parameters differs between the groups. We 
will also examine whether the decrease of the mitoPO2 precedes decreases of other measures of 
tissue oxygenation and microcirculation. 
5.1.2 Clinical Care 
It is standard clinical practice that most patients receive a central venous (vena jugularis, incidentally 
vena subclavia) and an arterial (arteria radialis) catheter when admitted to the intensive care unit. 
Patients with anaemia having an arterial catheter will be included in the study. During the pilot phase 
of the study, patients did not need to have a central venous catheter since the pilot study is about the 
feasibility of the mitoPO2 measurement and not the outcomes. Blood samples will be drawn from 
these catheters. Patient management will take place according to usual care. When patients meet the 
inclusion criteria, an ALA-patch will be placed. After at least four hours PpIX induction, the cutaneous 
mitoPO2 sensor will be placed to make the first measurements. Transfusion will be given in an one 
hour. The clinician can deviate from this time frame if the condition of the patient doesn’t permit a 1 
hour lead time. After red blood cell transfusion, measurements of the mitoPO2 will be made at 
predefined moments.  
 
5.2 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for urgent medical 
reasons. These subjects will not be subjected to follow-up. 
5.3 Data collection 
5.3.1 MitoPO2 measurements 
MitoPO2 is measured at the skin at the predefined moments described in detail in the next paragraph. 
With each measurement, the circumstances of the measurement will be recorded in the log (e.g. skin 
temperature) to assess possible bias of measurements. 
 
5.3.2 Gathering and handling of clinical data  
While waiting for the induction of PpIX after placement of the ALA patch, data will we gathered. 
Appendix 2 shows an overview of the data collection. First of all, demographic data will be collected: 
age in years, gender, weight, height, Body Mass Index, allergies, medication use, comorbidities (acute 
and chronic), and reason of ICU admission. Also, pulmonary function, renal function, APACHE-II and 
APACHE-IV will be assessed. The demographic data can be obtained from the hospital’s electronic 
patient dossier system (EPD).  
 

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Clinical data will be assessed as well, which are: heart frequency (HF), blood pressure (BP), central 
venous pressure (CVP), and peripheral oxygen saturation (SpO2). These will be assessed at the 
same time points as the mitoPO2 measurements. Daily the Glasgow coma scale, 24h-urine 
production, electrocardiogram (ECG), RIFLE classification, SOFA score, RASS, and ICDSC score will 
be collected. These measurements are already done, as part of the daily intensive care management, 
and will be collected using the EPD. Other data collected from the EPD are: vasopressor use, 
inotropic therapy, mechanical ventilation duration and settings, P/F ratio, Aa gradient, need for 
continuous renal replacement therapy (CRRT) and, if applicable, duration of CRRT. Also, cardiac 
index will be measured before and after red cell transfusion with the help of a non-invasive Vigilance 
II. This measurement isn’t part of standard care. 
5.3.3 Blood samples  
In addition to clinical characteristics and mitoPO2, we will measure Hb, haematocrit, arterial oxygen 
saturation (SaO2) and oxygen tension (PaO2), central venous oxygen saturation (ScvO2) if applicable 
and oxygen tension (PvO2) if applicable, and lactate in arterial and venous blood(if applicable) 
samples taken at the predefined moments before and after red cell transfusions. The collected blood 
will be immediately transported via the regular transport tubing system to the CKCL and CKHL for 
these analyses after being labelled with the unique subject number. Other measurements that will be 
done, in venous blood are: troponin, creatinine kinase, and creatinine. Urine will be collected for 24 
hours on the day of transfusion itself and the day after, to calculate the glomerular filtration rate (GFR). 
Blood samples are already taken multiple times during a 24-h ICU stay, as part of a normal intensive 
care management. For this study troponin is not part of the daily routing of the ICU. Therefore, 
determination of troponin will be added. Appendix 2 gives an overview of measurements part of 
standard care, and which of those measurements are part of the study. 
 
Information regarding ICU stay, hospital stay and mortality (ICU and hospital) will be available in and 
collected from the EPD. The follow up period will be three months. All data will be entered after 
validation in a study database for subsequent tabulation and statistical analysis. The data will be 
handled confidentially and anonymously. Patients re-admitted to the ICU and already participating in 
this study will only be followed according to the study protocol. Thus re-admitted patients will not be 
included for the second time. 
5.3.4 Sample handling and Measurements: 
Samples will be collected from 
- 
central venous catheter (if applicable) 
- 
arterial catheter 
 
In order to make comparisons between different compartments of the body, blood samples (arterial, 
central venous) and cutaneous samples will be collected at predefined moments. 

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These moments during the pilot study are: 
- Before transfusion (=T0) 
- At the end of each transfusion (=T1) 
- 30 minutes after T1 (=T2) 
- 60 minutes after T1 (=T3) 
- 180 minutes after T1 (=T4) 
- 24 hours after T1 (=T5) 
 
With the help of the pilot study, an overview of 
the measurements and parameters were made. 
With the help of this overview, decisions were 
made regarding the number of measurement moments (e.g. no need for measurement 15 minutes 
after red cell transfusion). Details about which parameters are determined at each time point during 
this pilot study are described in appendix 2. For the laboratorial measurements a total of 42ml of blood 
will be taken (10ml at T0 and T5, 4ml at T1 and 2, 6ml at T3 and 7.5ml at T4). As can be seen in 
appendix 2, most of the measurements are part of standard care which minimalizes the burden for the 
patient. 
 
Analysis of red blood cell transfusion on the intensive care unit at LUMC showed 69% of transfusions 
occurred during daytime (between 8.00hr and 18hr). The remaining transfusions were evenly 
distributed in the evening and night time. Since transfusions outside the normal daytime are mostly 
because of urgent medical reasons, no measurements will be done in the evening or night.  
 
Besides time of transfusion, analysis of previous ICU data showed that the mean duration elapsing 
between the decision of a non-emergency red cell transfusion and the start of the transfusion is 2-3 
hours. Thus, maximum of 2 hours delay for start of transfusion during this study is achieved. However, 
at no time red cell transfusion will be delayed if the patient is clinically in need of a transfusion in order 
to have enough time to induce PpIX. To ensure this, the clinician responsible for the patient, and not 
the researcher, has the final decision in the timing of the red cell transfusion. The delay of transfusion 
will be recorded to gain an insight in delay of transfusion. 
5.3.5 Other measurements 
Also, cardiac output will be measured continuously by limited invasive methods such as the 
Vigileo/FloTrac.27 This measurement is done with the help of an already placed arterial catheter. VO2, 
DO2 and oxygen extraction ratio will be calculated.  
Sublingual side stream dark field imaging is the only non-invasive validated instrument to evaluate the 
microvascular perfusion in critical ill patients.28,29 It will be used during this study to assess the 
microcirculation before and after red cell transfusion and compare this with the mitoPO2 
measurements.  
 
Figure3: Measurement MitoPO2 with the Comet 

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5.4 Study parameters 
 
5.4.1 Main study outcomes 
The primary endpoint is the change of mitoPO2 before and after red cell transfusion. This will be 
compared to change in traditional parameters used to measure oxygenation and oxygen balance 
(ScvO2, SaO2, PaO2, PvO2, CI, and lactate).  
 
5.4.2 Secondary outcomes 
Secondary study outcomes include: 
-  Value of mitoPO2 measurements in predicting (ischemic) organ damage. The following parameters 
for organ damage (that are routinely collected) will be assessed: lung (pO2/FiO2 ratio), heart 
(troponin, CK, an ECG, CI), renal (creatinine, urine production, glomerular filtration rate, RIFLE 
classification), brain (delirium, RASS) and SOFA score. More details about the timing of these 
parameters are given in appendix 2. 
- Microcirculatory value of mitoPO2 
- Duration of mechanical ventilation 
- Need for renal replacements therapy 
- Length of ICU stay 
- Length of stay in hospital 
- Hospital mortality 
- ICU mortality 
- 90-day mortality 
- Assessment the safety of mitoPO2 measurements 
 
- Delay of transfusion because of mitoPO2 measurements 
 
- Adverse and serious adverse events of the mitoPO2 measurements 
 
 
 

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6. Statistical consideration 
 
6.1 Power estimate 
Since no prior mitoPO2 study had been performed on critically ill patients, no data was available to 
calculate the sample size needed for the prospective cohort study.  
Therefore, we performed a pilot study with 20 subjects to assess feasibility of the mitoPO2 
measurement and to calculate a standard deviation needed for the sample size calculation. The 
statistical significance level will be set at the 5% level. The sample size will be calculated to achieve a 
statistical power of 90%. We know from our pilot study performed on 20 critically ill patients that the 
mean mitoPO2 is 67.9 mmHg with a standard deviation of 13 mmHg. A difference in mitoPO2 of 
5mmHg after red cell transfusion is deemed to be clinically relevant.  Thus, the sample size calculation 
would subsequently be the following: 
𝑍𝛼/2 = 1.96 (two tailed for p = 0.05) 
𝑍1−𝛽= 1.28 (for β = 0.10) 
𝜇= 72.9 𝑚𝑚𝐻𝑔 
𝜇0 = 67.9 𝑚𝑚𝐻𝑔 
𝜎= 15 𝑚𝑚𝐻𝑔 
 
𝑛= (𝜎
𝑧𝛼/2+𝑧1−𝛽
𝜇−𝜇0
)
2
= 189. With an expected 10% drop out rate, the corrected sample size will be 189 ∗
1.1 = 209 subjects. 
 
 
6.2 Statistical methods to be employed 
Absolute differences and 95% confidence intervals will be calculated. In addition to the univariate 
comparisons we will also calculate the differences for each of the parameters independent of the other 
parameters using multivariate models. In addition, we will perform similar analyses in subgroups of Hb 
and reason of admission (classified by APACHE IV) to assess whether the observed effect of 
transfusion on the parameters differs between the groups. We will also examine whether the decrease 
of the mitoPO2 precedes changes of other measures of oxygenation. 
 
For the dichotomous outcomes logistic regression will be applied. For time-to event outcomes the 
univariable analysis will be performed using the Kaplan-Meier method, the multivariable analyses the 
Cox proportional hazard model. To determine the discriminative power of the model, the ROC curve 
with the c-statistic. P < 0.05 will be considered statistically significant for the primary outcome 
measure. The statistical analysis will be conducted using the SPSS (Statistical Package for the Social 
Sciences) statistical package, release 23.0 (SPSS Inc., Chicago, IL) and STATA. 
 
 

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7. Safety reporting 
 
7.1 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and 
the reviewing accredited METC if anything occurs, on the basis of which it appears that the 
disadvantages of participation may be significantly greater than was foreseen in the research 
proposal. The study will be suspended pending further review by the accredited METC, except insofar 
as suspension would jeopardize the subjects’ health. The investigator will take care that all subjects 
are kept informed. 
 
7.2 Possible (serious) adverse events and suspected unexpected serious adverse reactions 
The ALA patches are already used in and approved for the therapy of benign skin disorders with 
Photodynamic Therapy (PDT). Its use is safe and provides good treatment results with excellent 
cosmetic outcome. The 2x2cm large Alacare patches (8mg) were developed by Photonamic and are 
marketed for the treatment of actinic keratosis, a benign skin disorder, with photodynamic therapy. In 
dermatology, its use has been investigated.10 In phototherapy, the treatment effect depends on the 
production of singlet oxygen by photoactivation of PpIX. Excitation of PpIX induces apoptosis in cells 
as a result of oxygen-radical formation. In general, this requires illumination with continuous light and a 
high cumulative light dose. In contrast, PpIX-TSLT uses short-pulsed excitation and a total light 
dosage, i.e., orders of magnitude less than used for photodynamic therapy.10 A recent systematic 
review showed no significant (serious) adverse effects of ALA patches.30  
 
Side effects of ALA described with photodynamic therapy are burning of the skin (<50%, resolved in 1-
4 days), burning sensation of the skin (<92%), pruritus, hyperpigmentation (22-36%), edematous 
lesion (35%), application site erythema (92-99%), local pain (1-92%, <30% severe), application site 
irritation (72%), local haemorrhage(1-4%), and headache (<10%). Most of these reactions resolved 
within 2 weeks. There are no reported (serious) adverse events.16,22,23 Since the light load at the 
cutaneous-PpIX-TSLT is much lower than in phototherapy, no adverse events are expected. While 
PpIX-TSLT is likely to be safe, the effects of phototoxicity after PpIX induction should always be 
considered a potential risk requiring a risk and safety assessment in any intended application.10 
7.3 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the study, 
whether or not considered related to the investigational product. All adverse events reported 
spontaneously by the subject or observed by the investigator or his staff will be recorded. 
 
7.4 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 

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- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity;  
- 
Any other important medical event that did not result in any of the outcomes listed above 
due to medical or surgical intervention but could have, based upon appropriate judgement 
by the investigator.  
An elective hospital admission will not be considered as a serious adverse event. 
 
The investigator will report all SAEs to the sponsor without undue delay after obtaining knowledge of 
the events. Patients on the ICU are critically ill and therefore more prone to SAEs. ICU mortality in the 
Netherlands is approximately 8.4%. Thirty-five percent of the patients are in need for vasopressor 
therapy, and 46% of the patients are mechanically ventilated. Furthermore, the National Intensive 
Care Evaluation Report of 2015 reports that the mean ICU admittance duration is 3 days with a mean 
in hospital admittance duration of 13 days. Around 80% of the total ICU patients have a low APACHE 
IV score, and therefore a mortality chance of less than 30%.31 Thus, SAEs in relation to underlying 
disease and expected SAEs during the ICU course will not be reported.  
 
The coordinating investigator will report the SAEs through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 7 days of first knowledge for SAEs that result in 
death or are life threatening followed by a period of maximum of 8 days to complete the initial 
preliminary report. All other SAEs will be reported within a period of maximum 15 days after the 
sponsor has first knowledge of the serious adverse events. 
 
7.5 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational product (in this 
case the ALA patch as part of the mitoPO2 measurement) related to any dose administered. No 
SUSARs of the ALA patch are reported. Therefore, no SUSARs are expected during this study. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 7.4); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the ALA patch, regardless of the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity of the 
adverse reaction are not in agreement with the product information as recorded in: 
- 
Summary of Product Characteristics (SPC) for the COMET device; 
- 
Investigator’s Brochure for the ALA patch. 
 
The coordinating investigator of LUMC will report expedited the following SUSARs through the web 
portal ToetsingOnline to the METC: 
 
SUSARs that have arisen in the clinical study that was assessed by the METC; 

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 
SUSARs that have arisen in other clinical studies of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the subjects 
involved in the clinical study that was assessed by the METC. 
 
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted once every 
half year to the METC. This line-listing provides an overview of all SUSARs from the ALA patch, 
accompanied by a brief report highlighting the main points of concern.  
The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as notification 
to the competent authority. 
 
The expedited reporting will occur not later than 15 days after the sponsor has first knowledge of the 
adverse reactions. For fatal or life threatening cases the term will be maximal 7 days for a preliminary 
report with another 8 days for completion of the report.  
 
The coordinating investigator of LUMC is responsible for documentation of the SUSAR in the report 
and ToetsingOnline.nl. The principal investigators in the participating centres will inform the 
coordinating investigator in <24 hours after the occurrence of the SUSAR. In the case of occurrence of 
2 or more SUSARs in 6 months’ time, the study team will discuss possible termination of the study. 
 
7.6 Annual safety report 
In addition to the expedited reporting of SUSARs, the coordinating investigator will submit, once a year 
throughout the clinical study, a safety report to the accredited METC. This will be combined with the 
annual progress report.  
This safety report consists of: 
 
a list of all suspected (unexpected or expected) serious adverse reactions, along with an 
aggregated summary table of all reported serious adverse reactions, ordered by organ system. 
 
a report concerning the safety of the subjects, consisting of a complete safety analysis and an 
evaluation of the balance between the efficacy and the harmfulness of the ALA patch. 
 
7.7 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. Depending 
on the event, follow up may require additional tests or medical procedures as indicated, and/or referral 
to the general physician or a medical specialist. SAEs need to be reported untill end of study within the 
Netherlands, as defined in the protocol  
 
7.8 Data Safety Monitoring Board 
 
This is a moderate risk study in which the side effect of ALA are well described. The side effects are 
based on studies with photodynamic therapy. Since the total lightload in photodynamic therapy is 
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expected.10,32 Indeed, studies using the ALA plaster for mitoPO2 measurement in Rotterdam reported 
no serious adverse events and no SUSARs.21,33,36 These studies were performed on healthy 
volunteers,21 surgical patients33 and patients with chronic anaemia.40 However, these studies reported 
local side effects of the ALA plaster consisting of erythema, pruritus and transient hyperpigmentation. 
All these effects were temporary and disappeared mostly within 1-2 weeks’ time. No local side effects 
or other adverse events were seen in our pilot study. To ensure the safety of our study participants, 
monitoring will take place during the study. Furthermore, the in- and exclusion criteria are optimised to 
minimize the risk for participants. Since safety is part of our secondary outcome, we will have attention 
for AEs, SAEs and SUSARs. With an independent expert on data safety monitoring board confirming 
the needlessness of a DSMB for the INOX ICU-2 study, no DSMB was initiated. 
 
 
 

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8. Ethical considerations 
 
8.1 Regulation Statement 
The study will be conducted according to the principles of the Declaration of Helsinki (accepted by the 
WMA General Assembly, 9 October 2004, Tokyo) and in accordance with the Medical Research 
Involving Human Subjects Act (WMO).  
 
8.2 Recruitment and Consent 
The protocol of this study will be submitted to the Medical Ethics Committee of the Leiden University 
Medical Centre. The study will not commence before formal approval has been granted.  
 
Since research has shown that approximately 10% of critically ill patients can make autonomous 
decision regarding research participation, most decisions will be made by their substitute decision 
makers, who are already overwhelmed by the medical information of the patient itself. Furthermore, 
studies have shown that obtaining contact with their substitute decision makers can be difficult in 7%, 
no decision maker exist in 6% of the cases. In addition, approximately half of the opportunities to 
recruit patients are missed because of workload and narrow time windows for inclusion. Difficulties in 
recruitment of eligible patient can lead to delayed identification of (in)effective treatment/diagnostics 
and limitation of generalizability. Alternative consent models are therefore needed to improve 
recruitment and minimize withdrawal.24,25 Therefore, we will firstask all patients admitted to the ICU, or 
their legal representatives if the patient is not able to give his/her consent, expected to stay at the ICU 
for longer than 24 hours for participation in our study. Patients admitted to the cardiothoracic 
department which will be admitted to the ICU post-operatively, will be asked for an informed consent 
on the day before the cardiothoracic operation. An information leaflet will be given and informed 
consent will be sought. Patients or their legal representatives will be told that a person can only 
participate in the study if he/she meets the inclusion criteria.  
 
However, if the patient or legal representative could not have been reached in time for written 
informed consent, while the patient met the eligibility criteria, patient will be included with deferred 
consent procedure. Informed consent will be sought within 48 hours after inclusion of the 
patient.Deferred consent is therefore only used in situations where it is not possible to ask informed 
consent, including the following:  lack of decision-making capacity of the patient due to illness severity, 
delirium, use of sedatives, analgesics and life-sustaining treatments; inability to contact the legal 
representative; overwhelming nature of medical information for the legal representative therefore 
influencing the capability of informed decision-making.24,34 aAt the moment the decision is made to 
administer a red cell transfusion, the self-adhesive ALA-patch will be placed, if the patient meet the 
eligibility criteria, and data collection for the study started.Due to logistics, the normal time elapsing 
between the decision of a non-emergency red cell transfusion and the actual administration is 2 to 3 
hours. Therefore, the maximal delay of a non-emergency transfusion will be 1 to 2 hours. To ensure 
improper delay of red cell transfusion due to the study, the clinician responsible for the patient, and not 
the researcher, has the final decision in the timing of the red cell transfusion. 

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If written informed consent was given, but retracted by legal representative or patient after 
participation, the patient will be excluded and data will no longer be used.  If patient or legal 
representative denies participation during the study, data collected until that time point will be used in 
the analysis. No further data will be collected.  
 
8.3 Objection by minors or incapacitated subjects 
Section 4, subsection 2, of the WMO stipulates that a legally incompetent adult cannot be forced to 
undergo a treatment or behave in a particular manner in the context of non-therapeutic research 
against his or her will. Subjects younger than 18 years are already excluded from the study. If there 
are adults, that are incapable of giving informed consent, a legal representative will be asked to give 
informed consent according to the GCP guideline. When the subject is able to give informed consent 
after being included in the study by consent of the legal representative and denies any further 
participation, the subject will be excluded from the study without any consequences. The data 
collected up to that point will be used for analysis. No additional data will be collected. 
   
8.4 Benefits and risk assessment, group relatedness 
The proposed study aims to optimize transfusion strategies in critically ill patients with anaemia. The 
measurements necessary to assess the defined study endpoints are not expected to negatively 
influence the result of treatment.  
 
8.5 Compensation for injury 
The Leiden University Medical Centre has a liability insurance which is in accordance with article 7, 
subsection 6 of the WMO.  
The LUMC (also) has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for Clinical Research 
in Humans of 23th June 2003). This insurance provides cover for damage to research subjects 
through injury or death caused by the study. 
1. € 650.000, -- (six hundred and fifty thousand Euro) for death or injury for each subject who 
participates in the Research; 
2. € 5.000.000, -- (five million Euro) for death or injury for all subjects who participate in the 
Research;  
3. € 7.500.000, -- (seven million and five hundred thousand Euro) for the total damage 
incurred by the organization for all damage disclosed by scientific research for the 
Sponsor as ‘verrichter’ in the meaning of said Act in each year of insurance coverage. 
The insurance applies to the damage that becomes apparent during the study or within 4 years after 
the end of the study. 
 
 

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9. Administrative Aspects and Publication 
 
9.1 Handling and storage of data and documents  
All patients will be addressed to with a random patient identification code, which will be generated  by 
CASTOR EDC, the electronic case report form. The codebook will be stored digitally, outside the 
investigator site file folder, with restricted access. Only the research team, monitor, accredited METC 
and IGJ will have access to this folder. The code will be safeguarded by the coordinating investigator. 
The codebook will be encrypted with a password. All data will be stored for further publication. All 
handling of personal data will comply with the EU General Data Protection Regulation (GDPR) and the 
Dutch Act on Implementation of the General Data Protection Regulation (in Dutch: Uitvoeringswet 
AVG).  Source data on the COMET device will be anonymous, it only contains date and time of 
measurement, it is not possible to enter patient name, personal number or date of birth. The COMET 
can only retrieve the data of the current/most recent measurement series until the user selects a new 
series or measurement spot. To store COMET data, it will be downloaded via an USB memory onto a 
computer memory conforming to the institution’s privacy guidelines and the USB memory will be 
erased. The COMET data will be shared anonymously with the manufacturer.  
 
 
9.2 Monitoring and Quality assurance 
The risks associated with this study are moderate for the subjects (more information can be found in 
chapter 10 and in annex 4). Therefore, only on-site monitoring is needed, which will happen in 
accordance of the NFU guideline of monitoring. Being labelled as a moderate risk study, the 
monitoring will be moderate to ensure the integrity and safety of the study participants. The  
monitorpool of Leiden University Medical Center, which are qualified for monitoring, will monitor this 
study. The monitorpoolis an independent group of data managers or research nurses, who aren’t 
involved in the study in any way other than monitoring. They will report to each principal investigator 
per site and to the coordinating investigator. The head of department of each site will be notified by the 
monitor, when the monitor notices frequent or substantial omissions.  
 
9.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the accredited METC 
has been given. All amendments will be notified to the METC that gave a favourable opinion. All 
substantial amendments will be notified to the METC and to the competent authority. Non-substantial 
amendments will not be notified to the accredited METC and the competent authority, but will be 
recorded and filed by the sponsor.  
 
9.4 Annual progress report  
The investigator will submit a summary of the progress of the trial to the accredited METC once a 
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included and numbers of subjects that have completed the trial, unexpected problems and 
amendments.  
 
9.5 End of study report  
The investigator will notify the accredited METC of the end of the study within a period of 8 weeks. 
The end of the study is defined as the 90th day after the last patients’ admittance to the ICU. In case 
the study is ended prematurely, the investigator will notify the accredited METC within 15 days, 
including the reasons for the premature termination. Within one year after the end of the study, the 
investigator/sponsor will submit a final study report with the results of the study, including any 
publications/abstracts of the study, to the accredited METC.  
 
9.6 Public disclosure and publication policy  
The study protocol and analysis plan will be published before start of the study on clinicaltrials.gov. 
The results of the study will find their way into (inter–) national scientific journals and guidelines.  
 
 

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10. Structured risk analysis  
 
In the following analysis we distinguish potential issues of concern related to the mitoPO2 
measurements 
 
a. Level of knowledge about mechanism of action 
The mechanism of action of mitochondrial oxygen tension measurements is well investigated since 2006 
and published in several articles.10,17-19,35  
Compared with other techniques to measure oxygen at the tissue level, oxygen-dependent quenching 
of delayed fluorescence of PpIX has some distinct advantages. It is quantitative, and, unlike oxygen 
electrodes, once calibrated it does not need recalibration at the time of usage. As opposed to oxygen 
saturation measurements based on near-infrared spectroscopy, the measurement site within the 
tissue is well defined. Therefore, the signal is not sensitive to changes in vessel density, like capillary 
recruitment. Furthermore, the technique relies on lifetime measurements instead of intensity 
measurements and therefore is highly insensitive to changes in tissue optical properties occurring, 
e.g., in case of venous congestion.10 
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar biological 
mechanism 
Topical application of ALA is registered for use of photodynamic therapy of skin lesions. Its use for 
measuring mitoPO2 in healthy volunteers has been proven to be safe in the finished study with protocol 
number NL37911.078.11 in which the PpIX-TSLT technique was tested on 30 healthy volunteers. All 
healthy volunteers experienced the skin preparation and measurements as non-problematic. Due to 
either the skin preparation or the ALA patch, 45% of the volunteers suffered from mild pruritus and/or 
erythema on the actual measurement day; these minor complaints were no longer present the day after 
the measurements. Only two volunteers had transient hyperpigmentation of the skin after the 
measurements, possibly due to premature exposure of the primed area to sunlight (against our advice). 
The hyperpigmentation was temporary and disappeared within one month. None of the volunteers 
sustained long-term skin damage, as established one month after the experiments.21  
 
It was also used in finished study with protocol number NL56686.091.16 in May and June 2016 at UMC 
St. Radboud in Nijmegen. This study induced endotoxaemia in human volunteers. Among other 
measurements, the COMET measurement system and Alacare were used to measure oxygen 
availability and consumption. ALA was applied on the evening before the endotoxaemia day. The study 
again confirmed the safety profile of the COMET’s measurement also during endotoxaemia. The 
measurements of the COMET identified change of mitochondrial function in vivo during human 
endotoxemia.36  
 
Other protocols use the same measuring technique: The running studies with Protocol nr: 
NL51187.078.14 (Photodynamic therapy study) and Protocol nr: NL51937.078.15 (Non-invasive 
monitoring of mitochondrial oxygen consumption and oxygenation (COMET): observational clinical 

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study) and NL55664.078.15 (MOTIFATE pilot, a study of blood transfusion and fluid administration in 
chronic anaemia).  
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human cell 
material? 
Mitochondrial oxygen tension research has been performed in cells and animals prior to this research 
proposal.13,14,32,37,38 PpIX-TSLT is one of the central techniques used in translational research in the 
Laboratory of Experimental Anaesthesiology of Erasmus Medical Center. 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
Not applicable 
 
e. Analysis of potential effect 
The PpIX-TSLT technique as well as the COMET have been investigated and have been proven safe, 
as demonstrated by studies on volunteers and patients. The possible effect of phototoxity after PpIX 
induction is a potential risk, but because the PpIX-TSLT uses short-pulsed excitation and total light 
dosage is much less than used in photodynamic therapy, this risk is considered to be very low.17,39 
Therefore we expect no effects of the measurement for the patients (aside from reported temporary mild 
discomfort like erythema and pruritus associated with the topical application of ALA).  
 
f. Pharmacokinetic considerations 
Not applicable 
 
g. Study population 
Critically ill patients on the ICU are vulnerable patients. According to GCP guidelines, a non-
therapeutical study can only take place if it has a likelihood of benefit for group represented by the 
subject. With the results of this study a potential phase 2 study can be performed to tailor the transfusion 
strategies in critical ill patients to reduce both over- and under- transfusion.  Besides the benefit for 
critically ill patients, this study entails moderate risk and burden for the subjects since it is a non-invasive 
measurement with no known serious adverse reactions. 
 
Some patients will have an altered consciousness which makes informed consent impossible in 
these patients. Therefore, we will seek informed consent from the legally authorised representative 
according to the GCP guidelines. 
 
To minimalize delay of transfusion, the ALA patch will be placed after the indication for red cell 
transfusion is made and informed consent is present. In case of situations outlined in chapter 8.2,  
informed consent will be sought as soon as possible after inclusion, but no longer than 48 hours after 
inclusion. If no written informed consent is achieved, subjects will be excluded from the study. The 
clinician responsible for the patient, not the researcher, makes the final decision in the timing of the red 
cell transfusion. 
 

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h. Interaction with other products 
None expected, see Alacare product information.16 
 
i. Predictability of effect 
No effect of the measurements is expected. Minor temporary skin lesions can occur, as seen in the 
healthy volunteers. With the COMET we expect less effect, due to progressive technique since the study 
in healthy volunteers, so the light intensity and total light dosage will be even less.  
 
j. Can effects be managed? 
No effects of the mitoPO2 measurements are expected, but when there is any indication of a skin lesion 
(e.g. unexpected pain during the measurements), the measurements can and will be stopped. 
 
 
 
 
 

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11. List of abbreviations  
 
AE 
 
Adverse Event 
ALA-patch 
 Cutaneous patch with 5-aminolevulinic acid 
AR 
 Adverse Reaction 
CCMO 
 Central Committee on Research Involving Human Subjects; in Dutch: Centrale  
 
 Commissie Mensgebonden Onderzoek 
CKCL  
Central clinical chemical laboratory 
CKHL  
Central clinical haematological laboratory  
CI 
  Cardiac index 
DO2 
 
Oxygen delivery 
DSMB   
Data Safety Monitoring Board 
EPD 
 Electronic patient dossier 
GCP 
 Good Clinical Practice 
GFR 
 Glomerular filtration rate 
IC 
 Informed Consent 
ICU 
 Intensive Care Unit 
METC  
 Medical research ethics committee (MREC); in Dutch: medisch ethische  
 
 toetsing commissie (METC) 
MitoPO2 
Mitochondrial oxygen tension 
NFU 
 
Nederlandse Federatie Universitair Medisch Centra 
PaO2  
Arterial oxygen tension 
PDT 
 
Photodynamic therapy 
PpIX 
 
Protoporphyine IX 
PvO2  
Venous oxygen tension 
RIFLE  
Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease  
classification 
(S)AE  
(Serious) Adverse Event  
ScvO2  
Central venous oxygen saturation 
SaO2  
Arterial saturation 
Sponsor 
The sponsor is the party that commissions the organization or performance of  
the research, for example a pharmaceutical company, academic hospital,  
scientific organization or investigator. A party that provides funding for a study  
but does not commission it is not regarded as the sponsor, but referred to as a  
subsidizing party. 
TSLT  
Triplet State Lifetime Technique (protoporphyrin IX-triplet state lifetime technique) 
VO2 
 
Oxygen consumption 
WBP 
 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WMO 
 Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch- 
 
 wetenschappelijk Onderzoek met Mensen 
 

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12. References 
 
1. Corwin HL, Gettinger A, Pearl RG, et al. The CRIT Study: Anemia and blood transfusion in the 
critically ill--current clinical practice in the United States. Crit Care Med 2004;32:39-52. 
2. Hebert PC, Wells G, Blajchman MA, et al. A multicenter, randomized, controlled clinical trial of 
transfusion requirements in critical care. Transfusion Requirements in Critical Care Investigators, 
Canadian Critical Care Trials Group. N Engl J Med 1999;340:409-17. 
3. Hebert PC, Carson JL. Transfusion threshold of 7 g per deciliter--the new normal. N Engl J Med 
2014;371:1459-61. 
4. Chohan SS, McArdle F, McClelland DB, Mackenzie SJ, Walsh TS. Red cell transfusion practice 
following the transfusion requirements in critical care (TRICC) study: prospective observational cohort 
study in a large UK intensive care unit. Vox sanguinis 2003;84:211-8. 
5. Retter A, Wyncoll D, Pearse R, et al. Guidelines on the management of anaemia and red cell 
transfusion in adult critically ill patients. British journal of haematology 2013;160:445-64. 
6. Murphy GJ, Pike K, Rogers CA, et al. Liberal or restrictive transfusion after cardiac surgery. N Engl 
J Med 2015;372:997-1008. 
7. Zheng Y, Lu C, Wei S, Li Y, Long L, Yin P. Association of red blood cell transfusion and in-hospital 
mortality in patients admitted to the intensive care unit: a systematic review and meta-analysis. Crit 
Care 2014;18:515. 
8. Holst LB, Carson JL, Perner A. Should red blood cell transfusion be individualized? No. Intensive 
care medicine 2015;41:1977-9. 
9. Vincent JL. Indications for blood transfusions: too complex to base on a single number? Annals of 
internal medicine 2012;157:71-2. 
10. Mik EG. Special article: measuring mitochondrial oxygen tension: from basic principles to 
application in humans. Anesth Analg 2013;117:834-46. 
11. Konrad FM, Mik EG, Bodmer SI, et al. Acute normovolemic hemodilution in the pig is associated 
with renal tissue edema, impaired renal microvascular oxygenation, and functional loss. 
Anesthesiology 2013;119:256-69. 
12. Balestra GM, Mik EG, Eerbeek O, Specht PA, van der Laarse WJ, Zuurbier CJ. Increased in vivo 
mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: 
mitochondrial inhibition as driver of cardiac failure? Respiratory research 2015;16:6. 
13. Harms FA, Bodmer SI, Raat NJ, Mik EG. Non-invasive monitoring of mitochondrial oxygenation 
and respiration in critical illness using a novel technique. Crit Care 2015;19:343. 
14. Harms FA, Voorbeijtel WJ, Bodmer SI, Raat NJ, Mik EG. Cutaneous respirometry by dynamic 
measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo. 
Mitochondrion 2013;13:507-14. 
15. Romers LH, Bakker C, Dollee N, et al. Cutaneous Mitochondrial PO2, but Not Tissue Oxygen 
Saturation, Is an Early Indicator of the Physiologic Limit of Hemodilution in the Pig. Anesthesiology 
2016;125:124-32. 

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16. Alacare 8 mg pleister voor cutaan gebruik. (Accessed 19-10-2016, 2016, at http://db.cbg-
meb.nl/ords/f?p=111:3:0:ATC:NO::P0_DOMAIN,P0_LANG,P3_RVG1:H,NL,113539.) 
17. Mik EG, Stap J, Sinaasappel M, et al. Mitochondrial PO2 measured by delayed fluorescence of 
endogenous protoporphyrin IX. Nat Methods 2006;3:939-45. 
18. Mik EG, van Leeuwen TG, Raat NJ, Ince C. Quantitative determination of localized tissue oxygen 
concentration in vivo by two-photon excitation phosphorescence lifetime measurements. J Appl 
Physiol (1985) 2004;97:1962-9. 
19. Mik EG, Ince C, Eerbeek O, et al. Mitochondrial oxygen tension within the heart. J Mol Cell Cardiol 
2009;46:943-51. 
20. Mik EG, Johannes T, Ince C. Monitoring of renal venous PO2 and kidney oxygen consumption in 
rats by a near-infrared phosphorescence lifetime technique. American journal of physiology Renal 
physiology 2008;294:F676-81. 
21. Harms F, Stolker RJ, Mik E. Cutaneous Respirometry as Novel Technique to Monitor 
Mitochondrial Function: A Feasibility Study in Healthy Volunteers. PLoS One 2016;11:e0159544. 
22. Braathen LR, Szeimies RM, Basset-Seguin N, et al. Guidelines on the use of photodynamic 
therapy for nonmelanoma skin cancer: an international consensus. International Society for 
Photodynamic Therapy in Dermatology, 2005. Journal of the American Academy of Dermatology 
2007;56:125-43. 
23. Reinhold U, Dirschka T, Ostendorf R, et al. A randomized, double-blind, phase III, multicentre 
study to evaluate the safety and efficacy of BF-200 ALA (Ameluz((R)) ) vs. placebo in the field-directed 
treatment of mild-to-moderate actinic keratosis with photodynamic therapy (PDT) when using the BF-
RhodoLED((R)) lamp. The British journal of dermatology 2016;175:696-705. 
24. Burns KE, Zubrinich C, Tan W, et al. Research recruitment practices and critically ill patients. A 
multicenter, cross-sectional study (the Consent Study). American journal of respiratory and critical 
care medicine 2013;187:1212-8. 
25. Coppolino M, Ackerson L. Do surrogate decision makers provide accurate consent for intensive 
care research? Chest 2001;119:603-12. 
26. Shieh G. Sample size requirements for the design of reliability studies: precision consideration. 
Behavior research methods 2014;46:808-22. 
27. Thiele RH, Bartels K, Gan TJ. Cardiac Output Monitoring: A Contemporary Assessment and 
Review. Critical Care Medicine 2015;43:177-85. 
28. Cerny V. Sublingual microcirculation. Applied Cardiopulmonary Pathophysiology 2012;16:229-48. 
29. Sadaka F, Aggu-Sher R, Krause K, O'Brien J, Armbrecht ES, Taylor RW. The effect of red blood 
cell transfusion on tissue oxygenation and microcirculation in severe septic patients. Annals of 
intensive care 2011;1:46. 
30. Gupta AK, Paquet M, Villanueva E, Brintnell W. Interventions for actinic keratoses. The Cochrane 
database of systematic reviews 2012;12:CD004415. 
31. Evaluation NIC. Jaarboek 20152016 June 2016. 
32. Harms FA, Bodmer SI, Raat NJ, Mik EG. Cutaneous mitochondrial respirometry: non-invasive 
monitoring of mitochondrial function. J Clin Monit Comput 2015;29:509-19. 

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33. Ubbink R, Bettink MAW, Janse R, et al. A monitor for Cellular Oxygen METabolism (COMET): 
monitoring tissue oxygenation at the mitochondrial level. J Clin Monit Comput 2017;31:1143-50. 
34. Luce JM. Is the concept of informed consent applicable to clinical research involving critically ill 
patients? Crit Care Med 2003;31:S153-60. 
35. Mik EG, Johannes T, Zuurbier CJ, et al. In vivo mitochondrial oxygen tension measured by a 
delayed fluorescence lifetime technique. Biophys J 2008;95:3977-90. 
36. Zwaag J, Wefers Bettink MA, Kox M, Mik EG. Change of mitochondrial function in vivo during 
human endotoxemia: preliminary data. Ned Tijdschr Anesthesiologie 2016;27:100. 
37. Harms FA, Bodmer SI, Raat NJ, Stolker RJ, Mik EG. Validation of the protoporphyrin IX-triplet 
state lifetime technique for mitochondrial oxygen measurements in the skin. Opt Lett 2012;37:2625-7. 
38. Harms FA, de Boon WM, Balestra GM, et al. Oxygen-dependent delayed fluorescence measured 
in skin after topical application of 5-aminolevulinic acid. J Biophotonics 2011;4:731-9. 
39. Peng Q, Warloe T, Berg K, et al. 5-Aminolevulinic acid-based photodynamic therapy. Clinical 
research and future challenges. Cancer 1997;79:2282-308. 
40. R. Ubbink, P.A.W. te Boekhorst, E.G. Mik. Novel technique to monitor effect of transfusion on 
mitochondrial oxygenation. Poster ISICEM 2017. 
 
 
 

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13. Appendix 1 
 
Background on the measurement 
 
Photonics Healthcare B.V. (PH) has developed an innovative non-invasive bedside monitoring system 
to measure Cellular Oxygen METabolism (the COMET). This device, for the first time, provides insight 
in the adequacy of oxygen supply and the actual oxygen demand. Available technologies only 
measure parameters in the blood, where oxygen is transported, but not where it is actually needed. 
The COMET measures availability and utilization of oxygen where oxygen is utilized: in the tissue 
cells. 
 
The COMET measurement system consists of three parts: 
1. The bedside COMET monitor: has a multi-touch screen that directly, in real time, displays the 
mitoPO2, the functional parameter of mitochondrial oxygen availability in mmHg. Additionally, signal 
quality and sensor temperature indication at the time of the most recent oxygen measurement are 
shown. The user can set the measurement interval and follow the reaction of cellular oxygen to 
various interventions, or perform a spot-check, a single measurement on a specific part of 
the skin of the situation at a particular time. A measurement series in a situation where pressure 
temporarily stops local blood flow can measure oxygen utilization. The device is a standalone unit that 
can be mounted on a moveable cart or used on a table. 
2. The COMET Skin Sensor: is optically and electrically connected with the COMET measurement 
device. The optical fibers transmit optical signals for tissue excitation and fluorescence detection. The 
Skin Sensor creates a diverging excitation pulse allowing a superficial optical measurement of the 
oxygen in the mitochondria of the cells in the epidermis in a circular area of about 5 mm2. 
Fluorescence light from the tissue is collected and transmitted via the detection fiber towards the 
detector system. The sensor also detects ambient light and the temperature inside the sensor. 
3. ALA patch and sensor fixation: The dermatologic agent 5-aminolevulinic acid (ALA) is applied to the 
patient skin. The sensor is fixated to the site with the help of a patch on which the skin sensor is 
placed. Simple ALA patches were developed by Photonamic and are marketed for photodynamic 
therapy, ALA crèmes are available from Galderma, photocure, DUSA and Biofrontera. This set up 
allows the physician to place the ALA-patch anywhere on the patient’s skin and, after four hours, place 
the sensor to locally determine the mitoPO2 level in the cells of the epidermis, which is then displayed 
on the monitor. Measurements can be performed every second for several minutes, or every few 
minutes for a period of 24 hours on the same spot of skin. After that, another location for the patch 
should be chosen. 
 
The measurement specifications:  
Photonics Healthcare’s COMET monitor measures absolute oxygen availability precisely and exactly 
where it is needed: in the mitochondria of the active cells. This provides doctors with quantitative 
insight in the availability and consumption of oxygen in their patients’ cells and hence the need for a 
blood transfusion. To determine the oxygen tension in the mitochondria, the COMET measures the 

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lifetime of the triplet state of protoporphyrin IX (PpIX), a precursor of haeme and hence haemoglobin. 
The discovery that PpIX exhibits a ‘glow-in-the-dark’ effect with an oxygen-dependent life time was 
published in a Nature journal; patents are awarded in Europe and the US and exclusively licensed by 
PH. The COMET probes the tissue with very short pulses of green light generated by a diode-pumped 
solid state laser. This light is brought to the site via an optical fibre. The COMET then observes the 
decay of the intensity of delayed fluorescence from the red spectrum of light collected from the tissue 
in the first milliseconds after the flash. Under normal conditions, PpIX is present in very low 
concentrations due to the presence of a negative feedback loop in which a product of PpIX, haeme, 
acts to inhibit the production of aminolevulinic acid (ALA), a substrate for PpIX. This can be overcome 
by the exogenous administration of ALA, which on its turn leads to higher concentrations of 
protoporphyrin IX in the mitochondria.1 ALA patches are already used in and approved for the therapy 
of benign skin disorders with Photodynamic Therapy (PDT). Its use is safe and provides good 
treatment results with excellent cosmetic outcome. The 2x2cm large Alacare patches (8mg) were 
developed by Photonamic and are marketed for the treatment of actinic keratosis, a benign skin 
disorder, with photodynamic therapy. 
 
The key specifications of the COMET sensor system are: 
-Detection range: 0 – 200 mmHg (arterial oxygen tension does not exceed 100 mmHg in normal 
physiologic situations). 
-Tolerance of Monitor: +/- 15%, at least +/- 3 mmHg, for signal quality >75%, which is similar or better 
than many clinical measurement systems such as cardiac output (+/- 30%), tissue oxygen (+/- 30%) or 
haemoglobin (+/-20%) 2,3. Repeatability and stability of the measurement is high and absolute 
accuracy will improve as more information about the new measurement becomes known but already is 
higher than for some current parameters used in the clinic (for example cardiac output). 
-Time to result: 1 second (for a prepared measurement location, i.e. placement of ALA-patch 4 hours 
previously to measurement). 
-Simple handling (one hour of training is sufficient to train any doctoral student, nurse or doctor) 
-All-in-one system, no need for other equipment or control mechanisms. 
 
Current stage of development: 
The COMET measurement system has been developed based upon applicable regulations, standards 
(Medical device directive 93/42EEC, IEC 60601 rev 3, ISO 13485) and has been tested in preclinical 
tests and volunteer studies. The key milestones are described below. 
 
The crucial step in the invention is the exploitation of the oxygen dependent optical properties of 
protoporphyrin IX. Because this substance is made in the mitochondria and can be measured on the 
skin, the problem of signal location that has made all earlier attempts to directly measure cellular 
metabolism unsuccessful is solved. First publication of the feasibility of the measurements was in 
2006 in Nature Methods.4 
 

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Subsequently the methods was tested in vivo on various organs,5-7 validated against existing 
technologies8,9 and showed the measurement could be used to measure oxygen consumption i.e., 
cellular energy turnover in a first human volunteer.10,11 Further studies demonstrated that cellular 
oxygen availability and consumption can safely be measured using ALA patches and COMET 
prototypes in a study in 30 human volunteers at Erasmus MC in Rotterdam.12 it was shown that 
information from the skin is representative for other parts of the body.13 From September 2014 until 
April 2015, Van Diemen et al. measured the increase of cellular oxygen consumption with the COMET 
prototype in skin of 28 human volunteers after 4 weeks of daily statin use (Simvastatin, a cholesterol 
lowering drug suspected of modifying mitochondrial function) and its partial reversal by additional 
intake of ubiquinol (Q10), which contributes to mitochondrial function.13 Next to the COMET the study 
also measured phosphocreatine recovery time after muscle exercise in 7-Tesla-31P-MR spectroscopy 
and mitochondrial membrane potential in white blood cells. The practically perfect correlation of the 
results in all groups is a strong validation of PH’s measurement against a very expensive and 
cumbersome technique. 
 
PH developed the COMET monitor, the first clinical monitor for cellular oxygen and conducted 
preclinical research with the help of partners, a Eurostars grant and investors.14,15 
 
In patients receiving ALA assisted photodynamic therapy and during neurosurgery oxygen levels were 
measured with the COMET. These demonstrate that the COMET can be used in patients and 
measurements are stable. The study in this project will take the first step towards the use of this 
technology for blood transfusion in critically ill patients. 
 
References 
1 Kennedy JC et al. Photodynamic therapy with endogenous protoporphyrin IX: basic principles and 
present clinical experience, J. Photochem. Photobiol. B 6 (1990) 43–148. 
 
2 Park SY et al. Accuracy of cardiac output measurements during off-pump coronary artery bypass 
grafting: according to the vessel anastomosis sites. Korean J Anesthesiol. 2012 May; 62(5): 423–428.  
 
3.Critchley LA, Critchley JA. A meta-analysis of studies using bias and precision statistics to compare 
cardiac output measurement techniques. J Clin Monit Comput. 1999;15:85–91 
 
4 Mik et al. Cellular PO2 measured by delayed fluorescence of endogenous protoporphyrin IX. Nature 
Methods 3: 939-945 (2006) 
 
5 Mik et al..In vivo cellular oxygen tension measured by a delayed fluorescence lifetime technique. 
Biophys. J. 95: 3977- 3990 (2008) 
 
6 Mik et al., Cellular oxygen tension within the heart. J. Moll. Cell. Cardiol. 46: 943-951 (2009)  

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7 Harms et al. Oxygen dependent delayed fluorescence measured in skin after topical application of 5-
aminolevulinic acid. J Biophotonics 4(10): 731-739 (2011) 
 
8 Bodmer et al. Microvascular and cellular PO2 simultaneously measured by oxygen-dependent 
delayed luminescence. J Biophotonics 5(2): 140-151 (2012) 
 
9 Harms et al. Validation of the protoporphyrin IX-triplet state lifetime technique for cellular oxygen 
measurements in the skin. Optics Letters 37: 2625-2627 (2012) 
 
10 Harms et al. Cutaneous respirometry by dynamic measurement of cellular oxygen tension for 
monitoring cellular function in vivo. Mitochondrion Oct (2012)  
 
11 Mik. Special article: measuring cellular oxygen tension: from basic principles to application in 
humans. Anesthesia Analgesia. 2013 Oct;117(4):834-46 
 
12 Harms et al. (manuscript submitted for publication) and Floor Harms, PhD thesis, Rotterdam 26 
Nov 2014 
 
13 Harms et al. Cutaneous cellular respirometry: non-invasive monitoring of cellular function. J Clinical 
Monitoring Computing. 2014 Nov 12. DOI 10.1007/s10877-014-9628-9 
 
14 Van Diemen et al. WMS Targeting Mitochondria Berlin, October 2015 
 
15 E!7294 COMET was awarded in 2012, Photonics Healthcare raised funds from investors including 
High-Tech Gr疨nderfonds in 2013. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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14. Appendix 2: Overview of measurements 
 
 
 
 
 
•Daily
•creatinin, Hb, Ht, lactate, SaO2, ScvO2, PvO2, PvCO2, PaO2, PaCO2
•ECG
•APACHE II and IV (at admission), SOFA, RIFLE
•Continu
•SpO2, HF, BP, CVD, UP, ICDSC, RASS, GCS  
•inotropic therapy, vasopressor use
•fraction of inspired oxygen, mechanical ventilation duration and 
setting, renal replacement duration and settings
Standard measurements
•troponin
•CK
Extra determinations
•mitoPO2 at T0-T5
•ScvO2, SaO2, PaO2, PaCO2, PvCO2, PvO2 and lactate at T1-4
•Hb measurement at T3
•Cardiac index measurement with Vigileo II at T0 and T5
•Microcirculation with sublingual SDF imaging at T0 and T5
Extra measurements
Hb <6.3 mmol/l
non-
emergency 
transfusion
ALA patch
>4 hrs
MitoPO2 
measurement 
+collection of 
other 
measurements 
and data
log of 
conditions
Red blood cell 
transfusion
MitoPO2 
measurement + 
collection other 
measurements 
at predefined 
moments
end of 
transfusion
30min
60min
180min
24hrs
Informed 
consent
written 
consent 
<48hr
Data collection 
up to 3 months 
after the first 
measurement
hospital and 
ICU 
mortality
hospital and 
ICU stay

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End of (each) 
transfusion 
(=T1) 
ALA patch ≥4 hrs (=T0) 
T2 
T3 
24hrs after T1 (=T5) 
Abbreviations: BP=blood pressure; CK= creatinine kinase; CI= cardiac index; CVP=central venous pressure; ECG= electrocardiogram; FB= fluid balance; 
FiO2= fraction inspired oxygen; GCS= Glasgow coma scale;  Hb= haemoglobin; HF=heart frequency; Ht= haematocrit;  ICDSC= intensive care delirium 
screening checklist; ICU=intensive care unit; NYHA= New York Heart Association class; PaO2= arterial oxygen tension; PvO2= venous oxygen tension; 
RASS=Richmond agitation- sedation scale; RIFLE= Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease classification ; SaO2= arterial 
saturation; ScvO2= central venous oxygen saturation; SDF= side stream dark field imaging; SOFA= sequential organ failure assessment; SpO2=peripheral 
oxygen saturation; UP= urine production. 
Blood sample T0: 
-creatinine 
-troponin 
-CK 
-Hb 
-Ht 
-lactate 
-SaO2 
-ScvO2 
-PaO2 + PvO2 
-PaCO2 + PvCO2 
Urine for 24 hours 
Blood sample T5: 
-creatinine 
-troponin 
-CK 
-Hb 
-Ht 
-lactate 
-SaO2 
-ScvO2 
-PaO2 
-PvO2 
Urine for 24 hours 
Blood sample on T1-T4: 
-lactate 
-SaO2 
-ScvO2 
-PaO2 
-PvO2 
 
On T3: 
- Hb 
 
On T4: 
- Troponin and CK 
Demographic data: 
-age and gender 
-height+ weight 
-allergies 
-comorbidity 
-medication use 
-smoking 
-reason ICU admission 
-NYHA class 
-APACHE II and IV 
-pulmonary function 
Clinical measures T0: 
-HF 
-BP 
-CVP 
-SpO2 
-GCS 
-UP+FB  
-ECG 
-RIFLE 
-SOFA 
-RASS 
-ICDSC score 
-CI 
 
-Sublingual SDF 
Collection measurements on T1-4: 
-HF 
-BP 
-CVP 
-SpO2 
-GCS 
-UP+FB 
-ECG 
-RASS 
 
Clinical measures T5: 
-HF 
-BP 
-CVP 
-SpO2 
-GCS 
-UP+FB 
-ECG 
-RIFLE 
-SOFA 
-RASS 
-ICDSC score 
-CI 
-Sublingual SDF 
•(duration of)mechanical ventilation, FiO2, P/F ratio, Aa 
gradient
•inotropical therapy
•vasopressor use
•renal replacement therapy
Collection of other 
clinical data
MitoPO2 measurements on T1-5 
Timeframe in hours
MitoPO2  
 
 
 
 
 
 
before transfusion
transfusion
after transfusion
T4 

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15. Appendix 3: Skin Pad 
 
1. Description 
The Skin Pad is a non-sterile, single use device to forcelessly hold a sensor in optical access 
on intact skin for no more than 24 hours. 
 
The fixation of a sensor with the Skin Pad is designed to provide a steady and reliable 
measurement and allow repeated or continuous measurements over longer time periods that 
do not require the user to hold the sensor of fix it with tape that might cause changes to the 
signal. 
 
The Skin Pad on the skin with a sensor attached is shown in Figure 1. 
 
The Skin Pad consists of three layers and a release liner on each side to protect the adhesive 
surfaces before use. 
 
The central layer is a thin poleurethane film, double coated with a silicone gel adhesive and 
an acrylic adhesive on the opposite side (Scapa Soft Pro 6051). The silicon adhesive layer 
covering the entire surface area of the “skin side” of the Skin Pad is covered with a yellow 
LDPE release liner (Figure 2). Once this release liner is removed the silicon adhesive makes it 
possible to attach the sensor on the desired location on intact skin and reposition it several 
times. The adhesive power of the silicon adhesive is designed for sensitive skin and is called 
“skin friendly”. 
 
The “sensor side” of the Skin Pad is covered with a white PE foam (Scapa 9868/824) except 
for a central part with the dimensions of the sensor where the acrylic adhesive is exposed 
and where the sensor should be attached. The exposed acrylic adhesive is covered with a 
white release liner with a lip for easy removal before the skin sensor is attached. 
 
Small notches on the side of the Skin Pad can assist in orientation. For a sensor with an active 
measurement area 14 mm from the end of the sensor, these notches indicate the location of 
the measurement area in the midline of a sensor attached inside the clear adhesive area 
surrounded by the white foam. The Skin Pad should then be attached to the skin so that the 
mid- line of the Skin Pad and the line connecting the notches intersect at the center of the 
desired measurement location. 
 
Between the film and the foam, not accessible to the user or patient, is a metalized white poly- 
propylene film fixed to the film with a medical-grade rubber adhesive (Bioflex Rx848P). This 
layer will largely block ambient light to reduce the noise for the measurements. 
 
 
Figure 1: Skin Pad on the arm fixating a 
sensor next to a measurement device  
 
Figure 2: Yellow release liner of 
Skin Pad 
 
Figure 3: Top view of sensor 
with white release liner 

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1.1 Biocompatibility 
All materials in the Skin Pad are biocompatible and tested according ISO 10993-1:2009 Bio- 
logical evaluation of medical devices. Part 1: Evaluation and testing in the risk management 
process. All materials of the Skin Pad are considered non-cytotoxic (grade 0, ISO 10993-5) 
and negligible irritant (irritation score 0, ISO 10993-10). 
 
1.2 Non-Sterile 
The Skin Pad is a non-sterile product to be used on intact skin. 
 
1.3 Usage 
The Skin Pad is a single use product. 
 
1.4 Material datasheets 
Double coated layer: 
SP_datasheet 
6051.pdf Foam layer: 
SP_datasheet 9868-
824.pdf 
Aluminum  layer: 
SP_ datasheet RX848P TDS  Format.pdf 
 
2. Intended use 
The Skin Pad is a non-sterile, single use device to forcelessly hold a sensor in optical contact 
to intact skin for no more than 24 hours. 
 
3. Device Class 
The Skin Pad is a Class I device according to MDD 93/42 Rule 1 and a non-invasive device 
according to the medical devices guidance ‘MEDDEV 2.4/1 Rev.9 Juni 2010’. CE mark is 
pending. 
 
4. History of Document and Responsibility 
 
Versio
Date 
Responsible 
Reason 
1 
17-12-2013 
Rinse Ubbink 
Initial creation 
2 
7 Oct 2015 
Rinse Ubbink 
Updated according to new  design 
3 
7-Oct-2015 
Michael Münker 
Updated  description, generic 
 
 
 

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16. Appendix 4: Monitor plan according to NFU-guidelines 
 
 
 
Moderate risk but intensive monitoring 
Monitor frequency 
3 visits per year per centre 
Patientflow1  
Inclusion speed and percentage of patients fallen out (e.g. 
withdrawn, loss to follow up) 
Trial Master File / Investigator 
File 
Presence and integrity of investigation file 
 
Informed consent 
25% 
In-/exclusion criteria 
25% of all subjects of each centre2 
 
Source Data  
Verification 
25% (Based at predefined list of variables, including primary 
endpoint, which are in clear relation to safety and validity of the 
study) 
SAE3 
100% of the total subjects for possible missed SAE. It includes 
verification of procedures as well.3 
Investigated product 
Control of instructions given to subjects. Also, control of 
delivery, distribution, storage, return, expiration date and timely 
order of the ALA patch 
Study procedure 
Control of presence of instructions needed for study procedure. 
If needed, facilities and equipment will be checked as well. An 
initiation visit will be performed in each centre before start of 
the study in that centre. 
Laboratory 
Control of certification of laboratory (GLP certification) 
Biological monsters 
Control of assemblage, labelling and storage 
 
1 Monitoring of patient flow regardless of the risk classification since inclusion speed going to slow can 
threaten completion of investigation. 
2 If subjects are included wrongly, all files of the centre involved with that will be reviewed and 
controlled.  
3 If the reporting and / or appropriate reporting of serious adverse events (SAE) is incomplete or 
incorrect, all files of that particular centre need to be checked, regardless of the degree of intensity of 
monitoring.