Characterisation of Endothelial Cells in Different Inflammatory Pathologies
Conditions: Sepsis, Vasoplegia
Sponsor: Royal Surrey County Hospital NHS Foundation Trust

Protocol Version 1.0 23 January 2024  
1 
 
 
 
 
 
 
J-wire Extraction Discovery Initiative 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sponsor 
Royal Surrey NHS Foundation Trust (RSFT) 
Funder 
University of Surrey Doctoral College 
GUTS FBC 
ESICM 
Protocol Version and Date 
1.0 23 Jan 2024 
Chief Investigator 
Dr Ben Creagh-Brown 
Statistician 
University of Surrey Bioinformatics core facility 
-2 

Protocol Version 1.0 23 January 2024  
2 
Signature Page  
The undersigned confirm that the following protocol has been agreed and accepted and 
that the Principal Investigator agrees to conduct the study in compliance with the approved 
protocol and will adhere to the principles outlined in the Declaration of Helsinki, the 
International Conference on Harmonisation Topic E6: Guideline for Good Clinical Practice 
(ICH GCP), any relevant SOPs, and other regulatory requirements. 
I agree to ensure that the confidential information contained in this document will not be 
used for any other purpose other than the evaluation or conduct of the investigation 
without the prior written consent of the Sponsor. 
I also confirm that I will make the findings of the study publicly available through publication 
or other dissemination tools without any unnecessary delay and that an honest, accurate 
and transparent account of the study will be given; and that any discrepancies from the 
study as planned in this protocol will be explained. 
 
 
For and on behalf of the Study Sponsor: 
Signature:  
...................................................................................................... 
 
 
Date: 
....../....../...... 
Name (please print): 
...................................................................................................... 
 
 
 
Position: 
...................................................................................................... 
 
 
 
 
Chief Investigator: 
 
Signature: 
...................................................................................................... 
 
 
Date: 
....../....../...... 
Name: (please print): 
...................................................................................................... 
 
 
 

Protocol Version 1.0 23 January 2024  
3 
Table of Contents 
Signature Page ........................................................................................................................... 2 
Table of Contents ....................................................................................................................... 3 
1. 
Glossary .............................................................................................................................. 5 
2. 
Contacts ............................................................................................................................. 6 
3. 
Protocol Summary ............................................................................................................. 7 
4. 
Lay summary ...................................................................................................................... 8 
6. 
Trial design ....................................................................................................................... 12 
6.1. 
Study procedures ...................................................................................................... 13 
Part 1, Cohort 1: Healthy volunteers ................................................................................... 13 
Part 1, Cohort 2: Surgical cohort .......................................................................................... 14 
Part 1, Cohort 3: Sepsis cohort ............................................................................................ 15 
Part 2, Cohort 1: Surgical cohort .......................................................................................... 16 
Part 2, Cohort 2: Sepsis cohort ............................................................................................ 17 
7. 
Eligibility criteria .............................................................................................................. 19 
7.1. 
Inclusion – Healthy volunteer ................................................................................... 19 
7.2. 
Exclusion – healthy volunteer ................................................................................... 19 
7.3. 
Inclusion – Surgical patients ...................................................................................... 20 
7.4. 
Exclusion – Surgical patients ..................................................................................... 20 
7.5. 
Inclusion – Critically ill patients ................................................................................. 20 
7.6. 
Exclusion – Critically ill patients ................................................................................ 20 
7.9. 
Recruitment and screening ....................................................................................... 21 
7.10. 
Consent .................................................................................................................. 21 
7.11. 
Baseline data and previous medical history .......................................................... 23 
7.12. 
Follow up ............................................................................................................... 23 
7.13. 
Withdrawal criteria ................................................................................................ 23 
7.14. 
End of study ........................................................................................................... 24 
8. 
Adverse events ................................................................................................................. 25 
8.1. 
Reporting procedures ............................................................................................... 26 
9. 
Statistics and data analysis .............................................................................................. 26 
9.1. 
Part 1 Sample size calculation ................................................................................... 26 
9.3. 
Planned recruitment rate .......................................................................................... 27 
9.4. 
Statistical analysis plan .............................................................................................. 27 

Protocol Version 1.0 23 January 2024  
4 
10. 
Data handling ................................................................................................................ 27 
10.1. 
Data collection tools and source document identification ................................... 27 
10.2. 
Data handling and record keeping ........................................................................ 28 
10.3. 
Access to data ........................................................................................................ 28 
10.4. 
Archiving ................................................................................................................ 29 
11. 
Monitoring, audit & inspection ..................................................................................... 29 
12. 
Ethical and regulatory considerations .......................................................................... 29 
12.1. 
Public and Patient Involvement ............................................................................. 29 
12.2. 
Co-enrolment ......................................................................................................... 29 
12.3. 
Protocol compliance .............................................................................................. 29 
12.4. 
Data protection and patient confidentiality .......................................................... 30 
12.5. 
Financial and other competing interests ............................................................... 31 
12.6. 
Indemnity ............................................................................................................... 31 
12.7. 
Dissemination policy .............................................................................................. 31 
12.8. 
Authorship eligibility guidelines ............................................................................ 31 
13. 
References .................................................................................................................... 31 
 

Protocol Version 1.0 23 January 2024  
5 
1. Glossary 
AE 
Adverse Event 
CRF 
Case Report Form 
DAMP 
damage-associated molecular pattern 
DAWN 
Disabilities and wellness network  
EC 
Endothelial cell 
ECBx 
Endothelial cell biopsy 
GCP 
Good Clinical Practice 
HTA 
Human Tissues Act 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICU 
Intensive care unit 
ISF 
Investigator Site File 
PAMP 
pathogen-associated molecular pattern 
PI 
Principal Investigator 
R&D 
Research, Development and Innovation department 
REC 
Research ethics committee  
RNA 
Ribonucleic acid 
RSFT 
Royal Surrey NHS Foundation Trust 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TMF 
Trial Master File 
UOS 
University of Surrey 
 
 

Protocol Version 1.0 23 January 2024  
6 
2. Contacts 
Chief Investigator 
Ben Creagh-Brown (bencb@nhs.net) 
Intensive Care Unit, RSFT 
Reader (Clinical), School of Biosciences and Medicine, 
University of Surrey 
Principal investigator 
Charlie Piercy (Charles.piercy@nhs.net) 
Research, Development & Innovation, RSFT 
Intensive Care Unit, RSFT 
 
PhD student, School of Biosciences and Medicine, University 
of Surrey 
Protocol Contributors 
Paola Campagnolo (p.campagnolo@surrey.ac.uk) 
Senior 
Lecturer 
in 
Cardiovascular 
Molecular 
Biology, 
University of Surrey 
Christian Heiss (c.heiss@surrey.ac.uk) 
Professor of cardiovascular medicine, University of Surrey 
 
Peer review 
Dr Ib Jammer, Haukeland University Hospital, Bergen, Norway 
Professor John Laffey, National University of Ireland 
Sponsor 
Royal Surrey NHS Foundation Trust 
Funder(s) 
University of Surrey  
GUTS FBC 
ESICM 
Statistician 
University of Surrey Bioinformatics core facility 
 

Protocol Version 1.0 23 January 2024  
7 
3. Protocol Summary 
Title 
Characterization of endothelial cells in multiple inflammatory 
pathologies 
Short title 
JEDI-2 
Sponsor  
Royal Surrey NHS Foundation Trust 
Design 
Prospective observational study of three cohorts: i) healthy 
volunteers, ii) surgical patients, iii) critically ill patients. 
Primary objectives 
To identify the changes in mRNA expression in endothelial cells from 
surgical patients and sepsis patients 
Secondary objectives 
Stratification of patients based on mRNA expression from 
endothelial cells. 
Characterisation of microparticles, histones and cell free DNA. 
Target accrual 
Part 1: 25 healthy volunteers, 20 patients with sepsis and 20 elective 
abdominal surgery 
Part 2: 20 patients with sepsis and 20 elective abdominal surgery 
Inclusion criteria (both parts) 
• ≥18 Years old 
• At RSFT for either and emergency admission to ICU, an 
elective surgery or as a healthy volunteer. 
Exclusion criteria (both parts) 
• Prisoners 
• Anticipated prohibitively difficult venous cannulation  
• Presenting with inflammatory diseases and disorders 
including but not limited to arthritis, peripheral artery 
disease, vasculitis, diabetes with end organ damage, 
cardiovascular disease and CKD. 
• Currently prescribed immunomodulatory medication or 
immunocompromised  
• Received chemotherapy within 2 weeks of predicted 
sampling.  
• 
History of recent trauma 
Number of sites 
1 
Duration of recruitment 
12-18 months  
Duration of patient follow-up Up to 28 days 
Definition of end of trial 
End of follow-up of last patient 

Protocol Version 1.0 23 January 2024  
8
4. Lay summary 
The immune system is a complex network of cells and molecules that protects the body from 
infection and injury. When the immune system is activated, it produces inflammation, which 
is a natural response to help heal the body. However, too much inflammation can be 
harmful and lead to serious complications, such as sepsis, low blood pressure, organ failure 
and death. 
The interaction of cells that line the blood vessels (endothelial cells, EC) with the immune 
system, is believed to be the root cause of these symptoms. When exposed to inflammation, 
the instructional molecules (RNA) inside the EC change. This leads to a change of operation 
promoting the severe symptoms previously mentioned. 
Researchers have developed new safe techniques to collect these cells from the blood 
vessels of patients to study disorders like diabetes, heart disease and stroke. This technique 
involves gently inserting a metal guidewire into an arm vein to collect ECs. 
This study plans to collect ECs from patients undergoing surgery or admitted to intensive 
care. We also plan to collect control samples from healthy volunteers. Samples will be 
collected over the duration of the patients to RSFT. The RNA will be removed from the cells 
and counted to highlight changes in instructions in the cells.  
Data from this study will potentially highlight new pathways involved in inflammation and 
help classify how some patients will react to current treatments. To obtain this data, this 
study will be split into 2 parts. Part 1 focuses on collecting one sample from a patient when 
they are at their most unwell states and comparing that to a sample from a healthy person. 
Part 2 will focus on key mRNA molecules identified during Part 1 and identifying how their 
expression changes over time. 
 
 
5. Background 
The immune response is a complex process that is essential for protecting the body from 
infection and orchestrating the repair of injured tissues. Activation can be caused by severe 
infection (sepsis), or from significant injury to tissues - such as occurs during major surgery, 
trauma, pancreatitis or burns(1,2).  
Pattern recognition receptors. The innate immune response can be triggered through 
pattern recognition receptors. In infection, these receptors detect pathogen-associated 
molecular patterns (PAMPs)(3) and in sterile tissue injury, damage-associated molecular 
patterns (DAMPs)(4). Profound activation can cause systemic inflammation and, if excessive, 
may be harmful. 
Sepsis and surgery. Sepsis is a dysregulated immune response to infection and is the most 
studied cause of systemic inflammation. It can be difficult to study due to its unpredictable 
nature and the uncertainty of defining the onset of infection(5). Planned major surgery 
causes similar systemic inflammation and many of its characteristic manifestations. 
Improving patients’ outcomes from surgery is important to study in itself, but surgery is also 
a good model for studying sepsis – with known time of onset and a predictable evolution (2). 

Protocol Version 1.0 23 January 2024  
9
The endothelium is a single layer of cells that lines the inside of blood vessels. It is 
constantly exposed to a variety of stimuli, including blood flow, shear stress, and 
inflammatory mediators, and it responds to these stimuli by secreting a variety of factors 
that regulate vascular tone, coagulation, and inflammation. It is also a selectively permeable 
barrier between the extravascular and intravascular compartments and can alter its 
permeability acutely(6–8). Altered endothelial cell (EC) function has predominantly been 
studied in chronic diseases (9,10) but is implicated in acute life-threatening conditions 
including sepsis and acute respiratory failure (for example, from COVID or other respiratory 
infections). Figure 1 shows our concept that ties injury to alterations in EC function and how 
this is clinically manifest. 
 
Figure 1: Conceptualised relationship between triggers of systemic inflammation, the 
immune response, the endothelial response, and the clinical features. 
New method – EC biopsy (ECBx). Researchers studying endothelial dysfunction in humans 
have access to a range of commercially available cells for in vitro studies, but there are 
substantial limitations to their use. Unprecedented insights could be gained from serial 
sampling of ECs from individuals with acute conditions. The first successful attempt to obtain 
ECs from patients was reported in 1999 by Feng et al (11). Their technique involved inserting 
a stainless-steel J-shaped guidewires into major vessels and collecting the tissue adherent to 
the guidewire. The technique has evolved into using other vascular implements such as 
endovascular coils and stents to study the effects of diverse diseases (10,12). Believing that 
ECs from patients would offer improved insights into the vascular alterations that take place 
during systemic inflammation, we performed a pilot study (IRAS 310961).  
We assessed the feasibility of ECBx in a surgical and intensive care setting. Discarded 
guidewires used during arterial and central line placements were collected from both 
surgical (n=16) and intensive care (n=16) patients. We also collected an additional biopsy 
from a peripheral arm vein in surgical patients only. ECs were then isolated from the 
guidewires and analysed using flow cytometry for absolute cell counts. From arterial lines 
we recovered an average of 536; from central lines we recovered an average of 598; from 
peripheral cannulation we obtained 736. One sample had RNA isolated which yielded a total 

Protocol Version 1.0 23 January 2024  
10 
of 105ng of material. We also assessed the tolerability of the ECBx through collection of 
adverse event (AE) data and a short questionnaire. There were no serious AEs related to 
ECBx, with only four patients (25%) of patients experiencing minor bruising which resolved 
without any complication. Patient feedback from the pilot study was overwhelmingly 
positive. All said that the biopsy was bearable and they could tolerate additional ECBx (mean 
of 3 additional biopsies). 
Biomarkers of EC function. It is not possible to directly measure the functions of the 
endothelium in patients but there are methods to gain insights. i) circulating levels of 
proteins reflect some aspects of EC function such as von Willebrand factor(13), 
Angiopoietin2(14) and angiotensin 2(15) ii) changes in circulating leukocytes (leukocytosis or 
leukopenia)(16) iii) micro particles released from EC can have pro-inflammatory, pro-
thrombotic and vasoregulatory properties and also contain microRNAs (miRNAs), non-coding 
RNA molecules regulating genes (17,18) iv) circulating miRNA (19) V) measuring aspects of 
EC structure and function, including flow cytometry.  
RNA sequencing of EC. We will process our patient-sourced ECs to preserve the mRNA for 
subsequent analysis. These analyses will provide insight into the EC function at the time of 
acquisition. RNA sequencing provides very detailed information about the relative 
abundance of many different transcripts. We can use knowledge of which transcripts are 
involved in which molecular pathways (for example a pro-inflammatory pathway like NFκB) 
to understand how putative pathways are expressed. Comparison of pathway expression 
between times within patient cohorts and between patient cohorts will allow us to see how 
EC respond in patients related to their clinical status.  
Hospital. The Royal Surrey hospital intensive care unit (ICU) receives >600 patients per year 
who have had major surgery that typically has been prolonged (>6 hours) and often 
associated with major tissue injury and blood loss (~1L). These patients have systemic 
inflammation and the predominant feature of altered EC function is loss of vasomotor tone 
causing hypotension. This is a major reason why they are managed on the ICU where we 
have specific expertise in treating hypotension. As an acute hospital we also regularly treat 
patients with severe infections and admit >300 patients/y with sepsis to the ICU. These 
patients will invariably have features of systemic inflammation and altered EC function. 
 
Research question 
Using the ECBx technique, our research question is: “In patients with sepsis or following 
major surgery, are changes in EC expression of putative pathways related to the clinical 
manifestations of SI?” 
 
Aims 
To determine the relationship between changes in EC expression in putative pathways, and 
the clinical manifestations and patient outcome for patients with sepsis or following major 
surgery.  
To identify the implicated pathways which will be useful to develop novel personalised 
therapies that may improve patient outcome. 
 

Protocol Version 1.0 23 January 2024  
11 
 
 
 
 
Objectives for Part 1 
• Recruit a cohort of healthy volunteers for method validation purposes. Undertake 
ECBx procedure (peripheral vein) to: 
o Optimise methodology for RNA extraction 
o Use as a control to compare with the patient groups. 
• Use a small number of these samples to develop methods of cell culture. 
• Concurrently recruit two patient cohorts: (A) planned major surgery, (B) acutely 
critically unwell patients (recently admitted to ICU with sepsis). 
• For each:  
o Undertake one ECBx procedure (peripheral vein). Isolate EC from guidewire. 
RNA sequencing on EC. In accordance with the corresponding schedule of 
events. 
o Characterise clinical status of the cohorts – for example extent of vasodilation 
as reflected in the dose of vasopressor drug being delivered to maintain an 
adequate blood pressure. 
o Characterisation of blood sample specifically microparticles, histones and cell 
free DNA, leukocyte activity. 
• Analyse RNA data to identify candidate gene expression changes that are linked to 
the change in phenotype  
 
Objectives for Part 2 
• Recruit a second cohort of patients with (a) sepsis and (b)those undergoing major 
abdominal surgery.  
• For each:  
o Undertake serial ECBx procedure (peripheral vein). Isolate EC from guidewire. 
RNA sequencing on EC. In accordance with the corresponding schedule of 
events. 
o Characterise clinical status of the cohorts – for example extent of vasodilation 
as reflected in the dose of vasopressor drug being delivered to maintain an 
adequate blood pressure. 
o Characterisation of blood sample specifically microparticles, histones and cell 
free DNA, leukocyte activity. 
• Use data from Part 1 to characterise the change in candidate gene expression. 
 

Protocol Version 1.0 23 January 2024  
12 
6. Trial design 
This is a two-part prospective observational cohort study comparing the mRNA changes in 
EC from two independent biological onsets.  
Part 1 focuses on identification of mRNA expression changes in one timepoint of acutely 
inflamed patients in comparison to a healthy control.   
Part 2 focuses on validating data from Part 1 in the same patient groups by analysing mRNA 
over several timepoints. 
 
Part 2 will commence upon completion of the Part 1 data review. See section 9 for further 
details.

Protocol Version 1.0 23 January 2024  
13 
6.1. 
Study procedures  
Part 1, Cohort 1: Healthy volunteers 
 
 
Screening 
Sample 
7-days Post sample 
Consent1 
X 
 
 
Demographics2 
X 
 
 
Medical history 2 
X 
 
 
ECBx3 
 
X 
 
Blood sampling3 
 
X 
 
AE and SAE monitoring4 
X 
X 
X 
 
 
1 - Consent must be performed to any study procedures. A standard consent or deferred consent model will be used, see section 7.9 for further 
details 
2 – Medical history and demographics will be collected from the available medical records on Surrey Safe Care and NHS SPINE 
3 – Severity of illness and clinical characterisation will use standardised methods such as qSOFA score and data from patient record to define. 
See section 7.16 for further details 
4 - Both samples to be collected at the same time. See section 7.15 for further details  
5 – See section 9 for further detail 
 
 
 

Protocol Version 1.0 23 January 2024  
14 
 
Part 1, Cohort 2: Surgical cohort 
 
Screening 
24 hours post-
surgery 
48 hours post-
surgery 
28-day follow-
up 
Consent1 
X 
 
 
 
Demographics2 
X 
 
 
 
Medical history 2 
X 
 
 
 
Severity of illness scoring3 
X 
X 
X 
 
ECBx4 
 
X 
 
 
Blood sampling4 
 
X 
 
 
Clinical characterisation3 
 
X 
X 
 
AE and SAE monitoring5 
X 
X 
X 
X 
Survival outcome 
 
 
 
X 
 
1 - Consent must be performed to any study procedures. A standard consent or deferred consent model will be used, see section 7.9 for further 
details 
2 – Medical history and demographics will be collected from the available medical records on Surrey Safe Care and NHS SPINE 
3 – Severity of illness and clinical characterisation will use standardised methods such as qSOFA score and data from patient record to define. 
See section 7.16 for further details 
4 - Both samples to be collected at the same time. See section 7.15 for further details  
5 – See section 9 for further detail 
 
 

Protocol Version 1.0 23 January 2024  
15 
Part 1, Cohort 3: Sepsis cohort 
 
 
Screening 
On admission 
24 hours post 
admission 
48 hours post 
admission  
28-day follow-up 
Consent1 
X 
 
 
 
 
Demographics2 
X 
 
 
 
 
Medical history 2 
X 
 
 
 
 
Severity of illness scoring3 
 
X 
X 
X 
 
ECBx4 
 
X 
 
 
 
Blood sampling4 
 
X 
 
 
 
Clinical characterisation3 
 
 
X 
X 
 
AE and SAE monitoring5 
X 
X 
X 
X 
X 
Survival outcome 
 
 
 
 
X 
 
1 - Consent must be performed to any study procedures. A standard consent or deferred consent model will be used, see section 7.9 for further 
details 
2 – Medical history and demographics will be collected from the available medical records on Surrey Safe Care and NHS SPINE 
3 – Severity of illness and clinical characterisation will use standardised methods such as qSOFA score and data from patient record to define. 
See section 7.16 for further details 
4 - Both samples to be collected at the same time. See section 7.15 for further details  
5 – See section 9 for further detail 

Protocol Version 1.0 23 January 2024  
16 
Part 2, Cohort 1: Surgical cohort 
 
Screening 
Pre-surgery 
24 hours post-
surgery* 
48 hours post-
surgery* 
28-day follow-
up 
Consent1 
X 
 
 
 
 
Demographics2 
X 
 
 
 
 
Medical history 2 
X 
 
 
 
 
Severity of illness scoring3 
X 
 
X 
X 
 
ECBx4 
 
X 
X 
X 
 
Blood sampling4 
 
X 
X 
X 
 
Clinical characterisation3 
 
X 
X 
X 
 
AE and SAE monitoring5 
X 
X 
X 
X 
X 
Survival outcome 
 
 
 
 
X 
 
1 - Consent must be performed to any study procedures. A standard consent or deferred consent model will be used, see section 7.9 for further 
details 
2 – Medical history and demographics will be collected from the available medical records on Surrey Safe Care and NHS SPINE 
3 – Severity of illness and clinical characterisation will use standardised methods such as qSOFA score and data from patient record to define. 
See section 7.16 for further details 
4 - Both samples to be collected at the same time. See section 7.15 for further details  
5 – See section 9 for further detail 
*Time and procedures subject to changed based on emerging data from part 1. See section 8 for further details 
 

Protocol Version 1.0 23 January 2024  
17 
Part 2, Cohort 2: Sepsis cohort 
 
 
Screening 
On admission* 
24 hours post 
admission* 
48 hours post 
admission * 
28-day follow-up 
Consent1 
X 
 
 
 
 
Demographics2 
X 
 
 
 
 
Medical history2 
X 
 
 
 
 
Severity of illness scoring3 
 
X 
X 
X 
 
ECBx4 
 
X 
X 
X 
 
Blood sampling4 
 
X 
X 
X 
 
Clinical characterisation3 
 
 
X 
X 
 
AE and SAE monitoring5 
X 
X 
X 
X 
X 
Survival outcome 
 
 
 
 
X 
 
1 - Consent must be performed to any study procedures. A standard consent or deferred consent model will be used, see section 7.9 for further 
details 
2 – Medical history and demographics will be collected from the available medical records on Surrey Safe Care and NHS SPINE 
3 – Severity of illness and clinical characterisation will use standardised methods such as qSOFA score and data from patient record to define. 
See section 7.16 for further details 
4 - Both samples to be collected at the same time. See section 7.15 for further details  
5 – See section 9 for further detail 
*Time and procedures subject to changed based on emerging data from part 1. See section 8 for further details

Protocol Version 1.0 23 January 2024  
18 
6.2. 
Study overview 
 
 
 
Figure 1: overview of the planned study. Part 1 – comparsion of an EC mRNA from 2 cohorts 
of patients taken at peak inflamation vs a healthy control. Data from this part will be 
reviewed to determine time points and genes of interest(see section 8 for further details). 
Part 2 - serial sampling looking at changes in gene expression from the genes of interest 
identified in Part 1.  

Protocol Version 1.0 23 January 2024  
19 
 
 
Figure 3: Schematic of major events for patients having planned major surgery. *Predicted 
time points for part 2 only. 
 
 
Figure 4: Schematic of major events for patients admitted to ICU with sepsis. *Predicted 
time points for part 2 only. **if required 
 
7. Eligibility criteria 
Eligibility criteria will be the same for both cohorts of patients in Part 1 and 2. Healthy 
volunteers eligibility criteria only applicable to Part 1.  
 
7.1. 
Inclusion – Healthy volunteer 
• Adult ≥ 18 years 
• Able and willing to give consent 
 
7.2. 
Exclusion – healthy volunteer 
• Not currently a patient within the hospital 
• Absence of inflammatory diseases and disorders including but not limited to 
arthritis, peripheral artery disease, vasculitis, diabetes, cardiovascular disease 
and CKD. 
• Not on immunomodulatory medications, such as corticosteroids 

Protocol Version 1.0 23 January 2024  
20 
• History of recent major trauma within the last 2 months (e.g., surgery or injury 
requiring hospitalisation)  
•  
 
7.3. 
Inclusion – Surgical patients 
• Adult ≥ 18 years 
• Patients admitted to RSFT for planned major surgery  
7.4. 
Exclusion – Surgical patients  
• Prisoners 
• Anticipated prohibitively difficult venous cannulation  
• Presenting with inflammatory diseases and disorders including but not limited to 
arthritis, peripheral artery disease, vasculitis, sepsis, diabetes with end organ 
damage, cardiovascular disease and CKD 
• Currently prescribed immunomodulatory medication or immunocompromised  
• Received chemotherapy within 2 weeks of predicted sampling 
• Receiving vasopressor support prior to surgery 
• History of recent major trauma within the last 2 months (e.g., surgery or injury 
requiring hospitalisation)  
 
7.5. 
Inclusion – Critically ill patients 
• Adult ≥ 18 years 
• Emergency admission to ICU at RSFT 
• Meets the sepsis 3.0 definition 
 
7.6. 
Exclusion – Critically ill patients 
• Prisoners 
• Anticipated prohibitively difficult venous cannulation  
• Presenting with inflammatory diseases and disorders including but not limited to 
arthritis, peripheral artery disease, vasculitis, diabetes with end organ damage, 
cardiovascular disease and CKD. 
• Currently prescribed immunomodulatory medication or immunocompromised  
• Received chemotherapy within 2 weeks of predicted sampling.  
• History of recent major trauma within the last 2 months (e.g., surgery or injury 
requiring hospitalisation) 
 
 
 
 
7.7. 
Justification for using healthy volunteers 

Protocol Version 1.0 23 January 2024  
21 
We are using healthy volunteers in this study to compare baseline mRNA expression to 
sepsis patients and surgery patients. This is because we want to isolate the effects of sepsis 
and surgery from other factors, such as the presence of other medical conditions or 
medications. Healthy volunteers are less likely to have confounding factors that could 
interfere with the results of our study. 
7.8. 
Incentivisation of healthy volunteers 
Staff at RSFT and staff and students at UOS were interviewed on how to best incentivise 
healthy volunteers’ participation and compensation for their time. A consensus agreement 
was reached suggesting that financial compensation would most appropriate. A median 
answer of £20 was suggested. 
Healthy volunteers will be remunerated £20 for their participation in this study.   
 
Trial procedures  
7.9. 
Recruitment and screening 
All patients will be identified from the electronic records systems at RSFT, by theatre lists or 
from referral by clinical staff. The study will use summary posters placed in staff areas to 
promote recruitment. 
Screening of patients will be done by checking clinical notes and from verbal confirmation 
from the participant. Patients who decline to participate or fail to meet eligibility will have 
the reason documented on the screening log. The PI or delegate can confirm eligibility. 
Healthy volunteers will be made up of members of staff and volunteers at RSFT and students 
at UoS. They will be identified by email advertisement from each individual organisation. All 
correspondence between volunteers and study team will be through the PI to maintain 
confidentiality. The wider study team will only be able to access anonymised date.  
 
7.10. 
Consent 
The PI retains overall responsibility for the conduct of research at their site, this includes the 
taking of informed consent of participants at their site. They must ensure that any person 
delegated responsibility to participate in the informed consent process is duly authorised, 
trained and competent to participate according to the ethically approved protocol, principles 
of GCP and Declaration of Helsinki. If delegation of consent is acceptable then details should 
be provided. 
Informed consent or agreement must be obtained prior to the participant undergoing 
procedures that are specifically for the purposes of the trial and are out-with standard 
routine care at the participating site. Personal consultees must be contacted first before 
approaching a professional consultee.  
The right of a participant to refuse participation without giving reasons must be respected.   
The participants are free to withdraw at any time from the trial without giving reasons and 
without prejudicing their further treatment and will be provided with a contact point where 
they may obtain further information about the trial. Data and samples collected up to the 

Protocol Version 1.0 23 January 2024  
22 
point of withdrawal will be used after withdrawal unless the participant has explicitly said so. 
The intention to utilise their data is outlined in the consent literature. Where a participant is 
required to re-consent or new information is required to be provided to a participant it is the 
responsibility of the PI to ensure this is done in a timely manner.  
The PI takes responsibility for ensuring that all vulnerable participants are protected and 
participate voluntarily in an environment free from coercion or undue influence. 
Where the participant population is likely to include a significant proportion of participants 
who cannot read or write, require translators, or have cognitive impairment, appropriate 
alternative methods for supporting the informed consent process should be employed. This 
may include allowing a witness to sign on a participant’s behalf (in the case of problems with 
reading or writing), or allowing someone to date the form on behalf of the participant, or 
providing Participant Information Sheets in a format easily understood by the participant 
population (in the case of minors or cognitive impairment). 
 
Cohorts: 
Healthy volunteers will be consented after the have shown an expression of interest in 
participating. They can be approached at any time to sign consent after they have registered 
their interest.  
Surgical patients will be consented prior to their surgery. They will be approached by a 
member of the clinical team to see if they would consider learning more about a potential 
research opportunity. If they are amenable, a member of the study team will introduce the 
study and present them with a PIS/ICF to review. This can be done prior to admission using 
telephone consent or in person on the day of surgery. There will be no deferred consent 
used. 
Sepsis patients will be recruited on admission to ICU. Should the patient have capacity, they 
will be approached by a member of the clinical team to see if they would consider learning 
more about a potential research opportunity. If they are amenable, a member of the study 
team will introduce the study to them and present the PIS. While the patient may have full 
capacity to make a decision, the anticipated stressful nature of an intensive care admission 
may leave them in a state where they do not want to read or sign a document. In this case a 
witnessed verbal agreement to participate will suffice until the patient is ready to sign the 
consent form. In patients who lack capacity, we will use a deferred consent model. A 
personal or professional consultee will be consulted. Once the patient has improved, the 
study team will then seek to be granted consent, retrospectively. If they do not regain 
capacity, we will still analyse their samples.  
Consent procedures can be completed by the PI or delegate. See figure 3 for visualisation. 

Protocol Version 1.0 23 January 2024  
23 
 
Figure 3 
 
7.11. 
Baseline data and previous medical history  
Information will be obtained from the medical notes following provision of consent by the 
participant. At time of enrolment we will collect demographic data, past medical history and 
current medical problems and current medications. Medical history and demographics in 
healthy volunteers will be collected as an oral history. The study will not look at medical 
records for this cohort. Data for patient cohorts will be collected from patient notes and 
from the summary care record from NHS SPINE. 
 
 
 
7.12. 
Follow up 
Follow up of patients will consist of obtaining their clinical status (Recovered/discharged 
from hospital/still in hospital/dead) from the patient’s clinical notes. This information is to 
capture any significant clinical events happening after discharge from ICU. No formal contact 
will be made with the patient, unless there are any outstanding AEs that require resolving. 
Follow up will take place at 28 days (±2 days) post enrolment.   
7.13. 
Withdrawal criteria 
It is within the remit of the treating physician to withdraw their patient from the study, if 
they believe that it is causing them significant harm or inappropriate to perform the 
procedure. The trial team will also withdraw patients from the study if the patient has 
suffered an serious adverse event (SAE) as a consequence of the trial procedures.  In both 
cases it must be explicitly documented in the eCRF the reason/s for withdrawal. Participants 
who are prematurely withdrawn may be replaced at the decision of the study team.  
It is the responsibility of the trial site to ensure that samples are appropriately labelled in 
accordance with the trial procedures to comply with the 1998 Data Protection Act. Biological 

Protocol Version 1.0 23 January 2024  
24 
samples collected from participants as part of this trial will be transported, stored, accessed 
and processed in accordance with national legislation relating to the use and storage of 
human tissue for research purposes and such activities shall at least meet the requirements 
as set out in the 2004 Human Tissue Act and the 2006 Human Tissue (Scotland) Act. 
7.14. 
End of study 
End of study will be defined as when the project has recruited all patients successfully, the 
last patient has completed follow-up and all adverse events have been resolved.  
 
7.15. 
Interventions 
ECBx is a technique that has been developed at RSFT for the safe and effective retrieval of 
EC for research. The procedure involves cannulating a peripheral arm vessel and gently 
passing a metal guide wire in to collect EC. The procedure is very low risk and very well 
tolerated in patients. AE data from our pilot study support this statement. Of the 16 
patients who received an ECBx, only 4 experienced minor bruising. All bruises resolved 
within a week of the procedure, did not require any additional treatment nor extend 
hospitalisation. No evidence of phlebitis or infection at the site of cannulation noted. More 
importantly the procedure did not restrict vascular access for standard of care. 
Participants in Part 1 will receive only 1 biopsy. 
Participants in Part 2 will receive up to 3 biopsies. 
Some patients may also receive arterial lines and central lines as part of their standard of 
care. These wires will be collected if available and used for in vitro modelling.  
 
A paired blood sample will be collected at the same time as the ECBx to look for biochemical 
markers of endothelial dysfunction, micro particles, Leukocyte function and circulating RNA. 
Serum and Plasma will also be collected and used to recreate the in Vivo phenotype, In Vitro.  
Participants in Part 1 will have 1 blood samples of 25 ml collected from a line or 
venepuncture at the same time as the ECBx. 
Participants in Part 2 will have 3 blood samples of 25 ml each collected from a line or 
venepuncture at the same time as the ECBx. 
Tissue samples, blood for leukocyte analysis, and serum and plamsa for in Vivo 
phenotyping will be sent to Charlie Piercy at the University of Surrey, where they will be 
prepared in room 35AY04 and then stored at -80oC in 37AY04. 
Serum for microparticle analysis and circulating RNA will be sent to University of Cologne 
Germany and covered in a separate MTA. 
 
 
 
 
 

Protocol Version 1.0 23 January 2024  
25 
7.16. 
Clinical characterisation and severity of illness scoring  
To assess severity of illness we will use qSOFA score and NEWS2. These assessments can be 
performed by looking at routinely collected information as part of standard of care and 
provide insight into how sick the patient currently is. 
For clinical characterisation we will look at the patients’ medical records. We will collect 
information on every aspect of their admission including but not limited to: 
• Blood – changes in blood biochemistry, whole blood counts, blood gasses, etc 
• Diagnosis – what the patient is initially diagnosed with and the change over the 
course of their admission 
• Targeted therapies – use of medications like noradrenalin to treat hypotension, 
inotropes to modulate heart contractions and antibiotics for infection 
• Concomitant medications – use of other medications that are not specifically given 
for the indication of sepsis or post-surgical hypotension. 
• MDT support – data from physiotherapy, dietetic and Speech and language therapy. 
 
7.17. 
Protocol deviations  
Major deviations from this protocol include mishandling of EC biopsy sample or 
blood, late processing of sample or incorrect storage of samples. These will be 
documented in the patients CRF and these samples may be excluded from analysis. 
Minor deviation from this protocol is any other deviation not listed above. This will 
be documented and use to describe any outliers to the data in the final analysis 
 
8. Progression to part 2 
Progression to part 2 will only take place after review of data and adverse events from part 1 
has been completed. The focus of the review will be to assess the variability of the data 
collected from part 1. If the variability is as predicted or less, no changes to the protocol will 
be made. If the variability is greater than expected, then the power will be recalculated. 
Based on the updated sample size, we will then reassess requirement for the current 
predicted sampling for part 2.  
 
Progression to part 2 will happen after an amendment to the protocol has been made 
confirming sample size requirement and sampling requirements.  
9. Adverse events 
All patients will be monitored in ICU as part of their routine clinical care pathway will 
potentially experience a range of serious events, including organ dysfunction and death. 
None of these events are plausibly related to the study and therefore they will not be 
reported. They will however, be recorded and included in the analysis of this study. 

Protocol Version 1.0 23 January 2024  
26 
For healthy volunteers, any adverse event related or non-related to the procedure will be 
recorded as an AE. Healthy volunteers will be followed-up at 1-week post procedure to 
confirm any adverse events. Any adverse events outstanding at a week post procedure will 
be followed-up until resolution. 
In those patients in whom we have undertaken EC samples using a guidewire in a peripheral 
vein, we will monitor for complications related to this procedure. Our collaborators 
(combined experience of >20 years using these techniques) do not report any serious 
adverse events related to the procedure. This is supported by the AE reporting from our 
pilot study, JEDI-1 (IRAS 310961). Nonetheless we will be vigilant specifically for: 
inflammation of the vein (phlebitis), thrombosis of the vein (thrombophlebitis), or infection 
around the vein or venous puncture site (cellulitis) for the 3 days following the procedure. 
 
9.1. 
Reporting procedures 
Adverse event data will be collected from the moment consent is signed until follow-up is 
completed. Adverse events will be reported twice a year to the study sponsor to 
demonstrate compliance with the regulatory guidelines.  
SAEs will be reported within 24 hours of the study team being made aware. SAEs will be 
reported to the sponsor and chief investigator who will report to the regulatory bodies. 
 
10. Statistics and data analysis 
10.1. 
Part 1 Sample size calculation 
Power analysis using simulations generated by the package ssizeRNA suggests that a sample 
size of 16 can detect a fold change of 2 with power 0.8 when 20,000 genes are tested at a 
false discovery rate of 0.05 if the proportion of non-differentially expressed genes is 90%, 
the average number of reads mapped per gene is 10 and dispersion 0.1. For part 1 we will 
recruit 20 patients per cohort to ensure that we get enough useable mRNA for further 
analysis.  We will also recruit 25 healthy volunteers, 20 for analysis and 5 to validate the 
method. 
 
 
 
 
 
 
 
 
 
 
 

Protocol Version 1.0 23 January 2024  
27 
 
 
 
 
10.2. 
Part 2 sample size calculation 
Using the sample size calculation method for a two-sample t-test with the provided 
parameters (effect size of 2, significance level of 0.05, power of 0.8, and a variability of 2) the 
analysis suggests that approximately 17 patients are needed. Variability is based on an 
expected result from part 1. Power analysis will be re-calculated after the part 1 data review. 
For Part 2, 20 patients per cohort will be recruited to ensure that data for analysis. 
 
 
10.3. 
Planned recruitment rate 
Scicluna et al, 2017 (Lancet), highlighted potential endotypes based on leukocyte 
transcriptomics. Of the four theoretical endotypes highlighted, the most common and most 
severe was prevalent in ~30% of the population(20). Data from the VOLUME CHASER study 
on the use of vasopressor in patients with septic shock also shows that up to 23% of patients 
will require high continuous administration of vasopressor(21).  
Only patients requiring organ support will be transferred to ICU. ICNARC data from Q1 2023 
for RSFT ICU showed that there were 22 high risk sepsis admissions (1.6 patients a week). 
There are over 20 major abdominal surgery cases a week admitted to ICU each week as part 
of their standard of care. 
Based on data collected from the pilot study we estimate to be able to enrol at a rate of 1.2 
patients a week. All patients will be recruited at one site by one member of the research 
team. Based on this we estimate that we will successfully recruit all patients within for 
part 1 within 12 months and part 2 within 8 months.  
10.4. 
Statistical analysis plan 
RNA extracted from endothelial cells will be sequenced and analysed using packages 
available on R. Preprocessing steps, including quality control, trimming, filtering, and 
alignment, will precede the analysis. Differential gene expression and pathway enrichment 
analyses will be performed using R packages such as DESeq2 and edgeR. The results will be 
presented through clear visualizations, such as heatmaps and volcano plots, providing a 
detailed overview of gene expression patterns. 
 
11. Data handling 
11.1. 
Data collection tools and source document identification 
ICH E6 section 1.51, defines source data as "All information in original records and certified 
copies of original records or clinical findings, observations, or other activities in a clinical trial 

Protocol Version 1.0 23 January 2024  
28 
necessary for the reconstruction and evaluation of the trial. Source data are contained in 
source documents (original records or certified copies)." 
The basic concept of source data is that it permits not only reporting and analysis but also 
verification at various steps in the process for the purposes of confirmation, quality control, 
audit or inspection. A number of attributes are considered of universal importance to source 
data and the records that hold those data. These include that the data and records are:  
• Accurate  
• Legible  
• Contemporaneous  
• Original  
• Attributable  
• Complete  
• Consistent  
• Enduring  
• Available when needed  
For this study, all data collected will be entered into an eCRF and all data stored digitally. All 
data will be collected on a tablet computer and stored centrally and securely on EDGE. The 
eCRF will be considered the source for all data in this study. Any data such as 
ultrasonography, ECGs or assessments that have physical sources will be translated into the 
eCRF.  These assessments will be available as a hard copy in the patient notes. 
11.2. 
Data handling and record keeping  
GCP requires that sponsors operating such systems validate the system, maintain SOPs for 
the use of the system, maintain an audit trail of data changes ensuring that there is no 
deletion of entered data, maintain a security system to protect against unauthorized access, 
maintain a list of the individuals authorized to make data changes, maintain adequate 
backup of the data, safeguard the blinding of the trial and archiving of any source data (i.e. 
hard copy and electronic). Data is transformed during processing, and it will be possible to 
compare the original data and observations with the processed data. Sponsors are 
responsible for ensuring compliance with the requirements outlined above when tasks are 
subcontracted. There should be no loss of quality when an electronic system is used in place 
of a paper system.  
 
11.3. 
Access to data 
Direct access will be granted to authorised representatives from the Sponsor, host 
institution and the regulatory authorities to permit trial-related monitoring, audits and 
inspections- in line with participant consent. 
Data can be freely shared with members of the collaborative group. Data may be shared 
with external researchers and industry if there is a consensus agreement by the 
collaborative group. This will documented in the PIS. 

Protocol Version 1.0 23 January 2024  
29 
 
11.4. 
Archiving 
Archiving will be authorised by the sponsor following publication of results. All trial related 
media and documents will be stored by Kelly’s in accordance with RSFT SOPs for a minimum 
of 5 years. Destruction of any essential documents will require authorisation from sponsor. 
12. Monitoring, audit & inspection 
Trial monitoring and protocol compliance will be led by R&D at RSFT. All visits will be 
conducted on-site by a delegate of R&D following RSFT SOPs. They will inspect the site files 
at initiation and close out of this study. They will also conduct a data quality check on the 
first patient/s recruited. If data collected is compliant with GCP and meets Trust standard, no 
further data quality checks will take place. 
The University of Surrey will be responsible for auditing protocol compliance with the HTA 
for all samples transferred to the Stag Hill Campus.  
13. Ethical and regulatory considerations 
13.1. 
Public and Patient Involvement 
As part of JEDI-1, we asked all patients involved in the study their opinions on multiple 
aspects of the study. The first area we focussed on was the consent procedure. We were 
concerned that the patients may feel they did not have enough time to make an informed 
decision given that they are usually consented within an hour of their procedure. All 26 
patients asked felt that they had enough time to make an informed decision and were not 
overwhelmed by our approach.  
Secondly, we appreciate that this is an invasive procedure and additional procedures may 
not be too welcome. We asked our patients if they would be happy to have an additional 
biopsy, and if yes how many additional biopsies could they manage. All patients said that 
they would be happy with an additional biopsy, with a mean of 3 additional biopsies – 4 in 
total. 
 
13.2. 
Co-enrolment 
Co-enrolment will be sought from any studies that are recruiting patients in relevant clinical 
areas. There are no observational studies that we will not co-enrol with, but we need to 
ensure that the other study teams do not mind co-enrolment. We will consider 
interventional trials on a case-by-case basis. All co-enrolment agreements will be stored in 
the site file. 
 
13.3. 
Protocol compliance  
Prospective, planned deviations or waivers to the protocol are not allowed must not be used 
e.g. It is not acceptable to enrol a participant if they do not meet the eligibility criteria or 
restrictions specified in the trial protocol 

Protocol Version 1.0 23 January 2024  
30 
Accidental protocol deviations can happen at any time. They must be adequately 
documented on the relevant forms and reported to the Chief Investigator and Sponsor 
immediately. Frequent deviations from the same root cause will be investigated to limit the 
effect it has on any data collected. 
Minor protocol deviations are attributed to events that have minimal impact on the data set. 
These include but are not limited too samples being taken outside of specified nominal 
timepoint, a non-delegated individual performing a task they are not assigned and variance 
in sampling depth. 
Major deviations are attributed to events that have a measurable impact on the data set and 
may necessitate a replacement participant. These include but are not limited too, enrolling a 
patient that is not eligible for the study, delayed transfer of sample to analytical laboratory 
and incorrect handling of sample in analytical laboratory. 
 
 
13.4. 
Data protection and patient confidentiality  
All investigators and trial site staff are compliant with the requirements of the Data 
Protection Act 1998 and General Data Protection Regulation (2018) with regards to the 
collection, storage, processing and disclosure of personal information and will uphold the 
Act’s core principles.  
All clinical data collected will be anonymised before it is shared with any members of the 
trial team that are not affiliated with the NHS or RSFT. Patients will receive a 3-letter code 
identifying which group they are in and a 3-digit number based on when they joined the 
study. The codes are: 
Part 1 
Surgical patients – SUR001-SUR020 
Critically unwell patients – ICU001-ICU020 
Healthy volunteers – HEL001-025 
Part 2 
Surgical patients – SUR0101-SUR120 
Critically unwell patients – ICU101-ICU120 
 
All personal data will be stored on RSFT EDGE clinical research platform. This validated 
system can be only be accessed by study team members within the NHS. The study will be 
further restricted so that only the team at RSFT will have access to any patient identifiable 
information.  
RSFT information governance and data protection team will monitor compliance of 
confidentiality and data protection. They are also responsible for any freedom of 
information requests and any complaints on data handling. 
All clinical information collected will be entered into a password secured eCRF compliant 
with ICH GCP E6 R2 (2018). The eCRF will be translated into a password protected document 

Protocol Version 1.0 23 January 2024  
31 
and shared via a private cloud space amongst the study team who will have read-only 
access. Data will be stored on the cloud space until the end of the project, then downloaded 
onto an encoded USB and stored for 15 years after the end of the study. Charlie Piercy will 
act as custodian of the data. 
13.5. 
Financial and other competing interests 
The study team have no financial or competing interest to declare. 
13.6. 
Indemnity 
As part of the sponsorship from R&D, this study will be placed under RSFTs indemnity 
policies. They will support the study and lead on any issues arising from this research 
ascertaining to indemnity and insurance. It is the responsibility of the trial team to notify the 
sponsor if any of these issues arise. 
13.7. 
Dissemination policy 
The data arising from this trial will be owned jointly between the study team named in this 
protocol. Upon completion of the study, the data will be analysed and written up in several 
formats. A copy of the full trial publication will be made available to both UoS, RSFT and 
GUTS FBC. We will work with DAWN at RSFT to create a lay summary and dissemination 
strategies to maximise the impact of our work with as many people as possible, including 
participants on the trial. We will also present the work as an abstract or presentation at 
conference with a focus on ESICM and EVBO. 
13.8. 
Authorship eligibility guidelines  
The study team named in the protocol will be named as authors on any subsequent 
publications arising from this work. However, the study team also acknowledge that there 
will be a lot of others who make contributions to the success of this work. Significant 
contributions which have a measurable outcome on the output of the project will be 
included in the authorship.  
 
14. References 
1. Boomer JS, Green JM, Hotchkiss RS. The changing immune system in sepsis. Virulence. 
2014 Jan 1;5(1):45–56.  
2. Bossy M, Nyman M, Madhuri TK, Tailor A, Chatterjee J, Butler-Manuel S, et al. The need 
for post-operative vasopressor infusions after major gynae-oncologic surgery within an 
ERAS (Enhanced Recovery After Surgery) pathway. Perioper Med. 2020 Sep 7;9(1):26.  
3. Gruda MC, Ruggeberg KG, O’Sullivan P, Guliashvili T, Scheirer AR, Golobish TD, et al. 
Broad adsorption of sepsis-related PAMP and DAMP molecules, mycotoxins, and 
cytokines from whole blood using CytoSorb® sorbent porous polymer beads. PLOS ONE. 
2018 Jan 25;13(1):e0191676.  
4. Pantalone D, Bergamini C, Martellucci J, Alemanno G, Bruscino A, Maltinti G, et al. The 
Role of DAMPS in Burns and Hemorrhagic Shock Immune Response: Pathophysiology 
and Clinical Issues. Review. Int J Mol Sci. 2021 Jan;22(13):7020.  

Protocol Version 1.0 23 January 2024  
32 
5. Jarczak D, Kluge S, Neirhaus A. Sepsis—Pathophysiology and Therapeutic Concepts. 
Front Med. 2021 May 14;8.  
6. Cahil PA, Redmond EM. Vascular endothelium – Gatekeeper of vessel health. 
Atherosclerosis.  
7. Dolmatova E, Wang K, Mandavilli R, Griendling K. The effects of sepsis on endothelium 
and clinical implications. Cardiovasc Res. 2021 Jan 1;117:60–73.  
8. Joffre J, Hellman J, Ince C, Ait-Oufella H. Endothelial Responses in Sepsis. Am J Respir Crit 
Care Med. 2020 Feb 26;202(3).  
9. Masaki N, Feng B, Bretón‐Romero R, Inagaki E, Weisbrod R, Fetterman J, et al. 
O‐GlcNAcylation Mediates Glucose‐Induced Alterations in Endothelial Cell Phenotype in 
Human Diabetes Mellitus. J Am Heart Assoc. 2020 Jun 6;9(12).  
10. Cooke DL, McCoy DB, Halbach VV, Hetts SW, Amans MR, Dowd CF, et al. Endovascular 
Biopsy: In Vivo Cerebral Aneurysm Endothelial Cell Sampling and Gene Expression 
Analysis. Transl Stroke Res. 2018 Feb 1;9(1):20–33.  
11. Feng L, Stern DM, Pile-Spellman J. Human endothelium: endovascular biopsy and 
molecular analysis. Radiology. 1999 Sep;212(3):655–64.  
12. Sheth SA, Verma A, Malhotra K, Tang X, Tateshima S, Jahan R, et al. Human Endothelial 
Cell Collection from the Middle Cerebral Artery in Acute Ischemic Stroke. J Stroke 
Cerebrovasc Dis. 2018 Mar 1;27(3):669–72.  
13. Rusu L, Minshall RD, Rusu L, Minshall RD. Endothelial Cell von Willebrand Factor 
Secretion in Health and Cardiovascular Disease. In: Endothelial Dysfunction - Old 
Concepts and New Challenges [Internet]. IntechOpen; 2018 [cited 2023 Oct 2]. Available 
from: https://www.intechopen.com/chapters/59581 
14. Akwii RG, Sajib MS, Zahra FT, Mikelis CM. Role of Angiopoietin-2 in Vascular Physiology 
and Pathophysiology. Cells. 2019 May 17;8(5):471.  
15. Birk M, Baum E, Zadeh JK, Manicam C, Pfeiffer N, Patzak A, et al. Angiotensin II Induces 
Oxidative Stress and Endothelial Dysfunction in Mouse Ophthalmic Arteries via 
Involvement of AT1 Receptors and NOX2. Antioxidants. 2021 Aug;10(8):1238.  
16. Farkas JD. The complete blood count to diagnose septic shock. J Thorac Dis. 2020 
Feb;12(Suppl 1):S16–21.  
17. Mesri M, Altieri DC. Endothelial cell activation by leukocyte microparticles. J Immunol 
Baltim Md 1950. 1998 Oct 15;161(8):4382–7.  
18. Reid VL, Webster NR. Role of microparticles in sepsis. Br J Anaesth. 2012 Oct 
1;109(4):503–13.  
19. Shu Z, Tan J, Miao Y, Zhang Q. The role of microvesicles containing microRNAs in vascular 
endothelial dysfunction. J Cell Mol Med. 2019 Dec;23(12):7933–45.  

Protocol Version 1.0 23 January 2024  
33 
20. Scicluna BP, Vught LA van, Zwinderman AH, Wiewel MA, Davenport EE, Burnham KL, et 
al. Classification of patients with sepsis according to blood genomic endotype: a 
prospective cohort study. Lancet Respir Med. 2017 Oct 1;5(10):816–26.  
21. Roberts RJ, Miano TA, Hammond DA, Patel GP, Chen JT, Phillips KM, et al. Evaluation of 
Vasopressor Exposure and Mortality in Patients With Septic Shock*. Crit Care Med. 2020 
Oct;48(10):1445.