Does the Release Profile of Nociceptin From Immunocytes Differ in Healthy Volunteers and Critically Ill Patients With Sepsis?
Conditions: Sepsis, Septic Shock, Sepsis Syndrome, Severe Sepsis
Sponsor: University of Leicester

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Does the release profile of nociceptin from immunocytes differ in healthy volunteers and critically ill patients with sepsis?  
Trial Protocol and Analysis 
 
Does the release profile of nociceptin from immunocytes differ in healthy 
volunteers and critically ill patients with sepsis? 
 
NCT03037281 
 
25/6/2018 
 
 
 

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Does the release profile of nociceptin from immunocytes differ in healthy volunteers and critically ill patients with sepsis?  
 
Full Study Title:  Does the release profile of nociceptin from immunocytes differ in healthy volunteers 
and critically ill patients with sepsis?  
Sponsor Reference No: 0554  
Ethics Ref: 16/EM/0046 Date and Version No: 25/06/2018 v2.0  
               
Chief Investigator:  
Professor David Lambert  
University Dept. Cardiovascular Sciences,  
Div. Anaesthesia, Critical Care and Pain 
Management, RK-CSB, Leicester Royal Infirmary, 
Leicester, LE2 7LX, UK.  
Tele: (+44) 0116 252 3161  
E-mail DGL3@le.ac.uk    
Investigators:   
Dr Christopher Hebbes, Prof Jonathan Thompson  
Sponsor:   
University of Leicester   
Funder:  
Biotechnology and Biological Sciences Research Council  
Royal College of Anaesthetists  
Signatures:  The approved protocol should be signed by author(s) and/or person(s) authorised to 
sign the protocol  
  
  
  
  
   
Confidentiality Statement  
All information contained within this protocol is regarded as, and must be kept confidential.  
No part of it may be disclosed by any Receiving Party to any Third Party, at any time, or in 
any form without the express written permission from the Chief Author/Investigator and / or 
Sponsor.  
  
  
Authors  
  
Professor David Lambert  
Professor Jonathan Thompson  
Dr Christopher Hebbes   
  
 

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Does the release profile of nociceptin from immunocytes differ in healthy volunteers and critically ill patients with sepsis?  
Signature Page  
  
Chief Investigator Name:  
___________________________________  
Chief Investigator signature: _________________________________ 
Date: _________________________________________________  
   
  
Sponsor Representative Name:  
________________________________  
Sponsor Representative signature: _____________________________  
Date:  
________________________________________________  
   
  
Principal Investigator Name: __________________________________  
Principal Investigator signature: ________________________________ 
Date: ________________________________________________  
   
(In cases of Multi-centre studies, this must be replicated for each site) 
TABLE OF CONTENTS  
To update table of contents (TOC), hover cursor over the top left hand corner until the whole TOC 
highlights.  Press the ‘F9’ button.  Choose ‘update entire table’.    
1.  AMENDMENT HISTORY ............................................................................................................ 6 
2.  SYNOPSIS .................................................................................................................................. 6 
3.  ABBREVIATIONS ....................................................................................................................... 9 
4.  BACKGROUND AND RATIONALE ........................................................................................... 10 
5.  OBJECTIVES ............................................................................................................................ 13 
5.1  Primary Objectives ............................................................................................................. 13 
5.2  Secondary Objectives ......................................................................................................... 13 
5.3  Exploratory End Point (where applicable) ........................................................................... 13 
6.  STUDY DESIGN ....................................................................................................................... 14 
6.1  Summary of Trial Design .................................................................................................... 14 

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6.2  Primary and Secondary Endpoints/Outcome Measures ...................................................... 14 
7.  TRIAL PARTICIPANTS ............................................................................................................. 16 
7.1  Overall Description of Trial Participants .............................................................................. 16 
7.2  Inclusion Criteria ................................................................................................................. 16 
7.3  Exclusion Criteria ................................................................................................................ 16 
8.  STUDY PROCEDURES ............................................................................................................ 17 
8.1  Informed Consent ............................................................................................................... 17 
8.2  Screening and Eligibility Assessment ................................................................................. 17 
8.3  Baseline Assessments ........................................................................................................ 18 
8.4  Randomisation and Codebreaking (if applicable) ................................................................ 18 
8.5  Subsequent Assessments .................................................................................................. 18 
8.6  Definition of End of Trial ..................................................................................................... 19 
8.7  Discontinuation/Withdrawal of Participants from Study ....................................................... 19 
8.8  Source Data ....................................................................................................................... 19 
9.  TREATMENT OF TRIAL PARTICIPANTS ................................................................................. 20 
9.1  Description of Study Treatment .......................................................................................... 20 
9.2  Storage of Study Equipment or Related apparatus ............................................................. 20 
9.3  Compliance with Study Treatment ...................................................................................... 20 
10. SAFETY REPORTING ............................................................................................................. 21 
10.1 Definitions ......................................................................................................................... 21 
10.2 Reporting Procedures for All Adverse Events .................................................................... 21 
11. STATISTICS ............................................................................................................................ 21 
11.1 Description of Statistical Methods ...................................................................................... 21 
11.2 The Number of Participants ............................................................................................... 21 
11.3 The Level of Statistical Significance .................................................................................. 22 
11.4 Criteria for the Termination of the Trial .............................................................................. 22 
11.5 Procedure for Accounting for Missing, Unused, and Spurious Data ................................... 22 
11.6 Procedures for Reporting any Deviation(s) from the Original Statistical Plan ..................... 22 
11.7 Inclusion in Analysis .......................................................................................................... 22 

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12. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ............................................................ 22 
13. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES ...................................... 23 
14. CODES OF PRACTICE AND REGULATIONS......................................................................... 24 
14.1 Ethics ................................................................................................................................ 24 
14.2 Sponsor Standard Operating Procedures .......................................................................... 24 
14.3 Declaration of Helsinki ....................................................................................................... 24 
14.4 ICH Guidelines for Good Clinical Practice ......................................................................... 24 
14.5 Approvals .......................................................................................................................... 24 
14.6 Participant Confidentiality .................................................................................................. 24 
14.7  Other Ethical Considerations ............................................................................................ 24 
15. DATA HANDLING AND RECORD KEEPING........................................................................... 24 
16. STUDY GOVERNANCE .......................................................................................................... 26 
16.1 Trial Steering Committee (TSC)......................................................................................... 26 
17. FINANCING AND INSURANCE ............................................................................................... 27 
18. PUBLICATION POLICY ........................................................................................................... 27 
19. REFERENCES ........................................................................................................................ 28 
20. APPENDIX A: STUDY FLOWCHART ...................................................................................... 31 
21. APPENDIX B: SCHEDULE OF PROCEDURES ...................................................................... 32 
 
  
  
  
 

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1. 
AMENDMENT HISTORY  
Amendment  
No.  
Protocol 
Version 
No.  
Date 
issued  
Author(s) of changes  
Details of Changes 
made  
1  
0.2  
07/12/2015  Hebbes  
Version number and  
title correction  
2  
1.1  
19/2/16  
Hebbes  
  
3 2.0 25/6/18 
Hebbes 
Single 
blood sample  
of 30mls (replacing 
2 
separate 
samples)  
Amendments 
to 
PIS and consent 
form  
Amendment to cell 
types examined  
(basophils 
removed)  
Sepsis screening 
criteria updated to 
Sepsis-3  
Updated 
sample 
sizes 
(reduced 
from 20 to 8)  
Updated sampling 
timeframe; 
increased to within 
48 hours of ITU 
admission  
Updated 
planned 
study end date to 
December 2018  
Updated 
secondary 
objectives – Not 
possible 
to 
measure 
N/OFQ 
levels using this 
assay as stated.  
Immune 
cell 
expression of N/OFQ 
and NOP by PCR as  
a surrogate  
2. 
SYNOPSIS  
  

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Study Title  
Does the release profile of nociceptin from immunocytes differ in 
healthy volunteers and critically ill patients with sepsis?  
Internal ref. no.  
0554  
Trial Design  
Case control  
Trial Participants  
Healthy volunteers  
Critically ill septic patients  
Planned Sample Size  
8 healthy volunteers  
8 critically ill septic patients  
Follow-up duration  
From admission to critical care, until discharge to ward based case or 
death  
Planned Trial Period  
January 2016 – December 2018  
Primary Objective  
To determine differences in the release profile of NOFQ from immune 
cells in the blood (neutrophils, eosinophils) between patients with 
sepsis admitted to the intensive care unit and healthy volunteers  
  
Secondary Objectives  
Determine differences in expression of N/OFQ and NOP genes in 
immune 
cells 
from 
septic 
and 
healthy 
volunteers 
using 
immunofluorescence and PCR as surrogates  
  
  
  

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Primary Endpoint  
The release of NOFQ will be determined by fluorescence of Chinese 
Hamster Ovary cells, expressing NOP, coupled to Calcium release and 
stained with a calcium sensitive fluorescent dye.  Relative fluorescence 
(change / basal) will be measured following layering of immune cells, and 
degranulation.  A relative fluorescence of >1.6 indicates a response, and 
the presence of NOFQ.  Measures will be number of “responder” cells  
 
Quantitative evaluation of N/OFQ and NOP expression in 
eosinophils and neutrophils, with a comparison between septic and 
healthy volunteers  
Secondary Endpoints  
Cell count and differential proportion of immune cell populations  
Physiological scores  
Mortality in-hospital, at 30 days  
Length of ITU stay, and hospital stay  

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3. 
ABBREVIATIONS  
AE  
Adverse event  
AR   
Adverse reaction  
BP  
Blood Pressure  
CI  
Chief Investigator  
CRA  
Clinical Research Associate (Monitor)  
CRF  
 
Case Report Form  
CRO   
Contract Research Organisation  
CT  
 
Clinical Trials  
EC  
 
Ethics Committee (see REC)  
EDTA  
 
Ethylenediaminetetraacetic acid  
GCP  
Good Clinical Practice  
GP  
General Practitioner  
GTAC  
Gene Therapy Advisory Committee  
ICF  
Informed Consent Form  
ISF  
Investigator Site File  
NHS  
National Health Service  
N/OFQ  
Nociceptin / Orphanin FQ  
NOP  
Nociceptin receptor  
NRES  
National Research Ethics Service   
PI  
Principal Investigator  
PIL/S  
Participant/ Patient Information Leaflet/Sheet  
R&D  
NHS Trust R&D Department  
REC  
Research Ethics Committee  
SAE  
Serious Adverse Event  
SAR  
Serious Adverse Reaction  
SIRS  
Systemic Inflammatory Response Syndrome  
SOP  
Standard Operating Procedure  
SUSAR  
Suspected 
Unexpected 
Serious 
Adverse 
Reactions  
TMF  
Trial Master File  
WCC  
White Cell Count  
  
  

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4. 
BACKGROUND AND RATIONALE  
Sepsis is defined as an overwhelming systemic response to infection (of any source), characterised 
by a systemic inflammatory response syndrome (SIRS), combined with suspicion, or evidence (from 
microbiological samples) of infection(Dellinger et al., 2013).  Classification is according to organ 
dysfunction, as septic shock (circulatory failure, in the presence of sepsis), severe sepsis (liver, 
kidney, circulatory, respiratory or gastrointestinal failure in the presence of sepsis), or sepsis (system 
inflammatory response in the presence of infection).  The mortality increases proportionately to the 
degree of sepsis, and, untreated is 40-50%.    
  
The underlying organism triggering the sepsis has a role in determining the severity; and early 
treatment with appropriate antibiotics reduces mortality dramatically in a time dependent 
manner(Kumar et al., 2006).  However, the control and the regulation of the immune system in the 
face of sepsis may also be implicated in survival.  
  
Following the discovery and cloning of the fourth opioid receptor (NOP)(Wang et al., 1994), the use 
of reverse pharmacology led to the sequencing of the endogenous ligand Nociceptin(Meunier et al., 
1995) and OrphaninFQ(Reinscheid et al., 1995) (N/OFQ).  It has a role in the regulation of the pain 
response and reward behaviours, and is increasingly considered as a therapeutic target in sepsis.  
Opportunities to regulate the detrimental effects of immune system over-activation in sepsis would 
potentially improve outcomes for this patient group.  
  
Evidence for the role of N/OFQ in the immune system  
  
Clinical studies in humans have demonstrated increased levels of NOFQ in sepsis(Stamer et al., 
2011; Williams & Thompson, 2008), following cardiopulmonary bypass(Thompson JP et al., 2013), 
and arthroscopy(Lee & Jeon, 2013).  Administration of a NOP antagonist in the caecal ligation model 
of septic peritonitis in the rat was improved mortality.  
  
There is evidence for the presence of N/OFQ and NOP in cells of the immune system at the 
molecular level.  Binding studies are inconclusive, with some suggesting the presence of NOP on 
immune cells(Peluso, Gaveriaux-Ruff, Matthes, Filliol, & Kieffer, 2001), whereas other authors have 
failed to find any evidence of NOP binding (Williams et al., 2007).    
Genetic studies have demonstrated the presence of NOP mRNA in human lymphocytes(Wick, 
Minnerath, Roy, Ramakrishnan, & Loh, 1996), monocytes(Peluso et al., 1998), and polymorphs(Fiset, 

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Gilbert, Poubelle, & Pouliot, 2003).  This discrepancy may be due to differences in functional, isolated 
human cells, versus the use of cloned human cell lines, or may suggest that NOP mRNA exists but 
not functionally transcribed, or may be upregulated in sepsis.  
  
mRNA for N/OFQ, and its peptide precursor pp-NOFQ has been demonstrated consistently in human 
splenocytes and leukocytes(Nothacker et al., 1996), lymphocytes(Arjomand, Cole, & Evans, 2002), 
and neutrophils(Fiset et al., 2003).  Whilst mRNA is demonstrated in PCR studies, the functional 
expression has not been evaluated, nor the identity of the releasing cell.    
  
Neither the significance nor source of nociceptin in sepsis is known.  The measurable rise in plasma 
concentrations following septic or inflammatory stimuli may represent a consequence or cause of 
the underlying septic response, and may therefore represent a therapeutic intervention, or a 
biomarker for sepsis severity, or prognostication.    
  
Risks and benefits  
This study requires a single 30ml blood sample to be taken from septic patients, within 48 hours of 
initial admission to the intensive care unit, into an EDTA bottle, stored at 18-22 degrees centigrade 
and processed within 2 hours of sampling.  A member of the investigative team competent at 
phlebotomy will collect these samples, either by venepuncture or by sampling from an indwelling 
arterial or central venous line using a standard aseptic non touch technique.  
  
A single 30 ml sample will be collected by venepuncture from healthy volunteers.  
The collection of this small volume of blood would be an additional procedure to the patient’s care.  
However, collection of this small volume of blood would not be anticipated to have any deleterious 
effect.  In order to avoid harm, data regarding allergies, medical problems and medication will be 
collected.    
  
Patient population  
The patient population for this study will be patients admitted to the intensive care unit with a 
diagnosis of sepsis.  Healthy volunteers will be recruited from staff and students within the 
department of Cardiovascular Sciences at the University of Leicester.  
  

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There is a likely role for NOP and NOFQ in the regulation of the immune system in sepsis.  Elevated 
plasma NOFQ in inflammatory conditions may be a cause or consequence of immune system 
activation, and could provide a new therapeutic target.  This is the first assay developed to 
demonstrate NOFQ release from single cells.  
Using this assay, we will determine the differences in functional NOFQ expression in immunocytes 
from healthy volunteers and septic patients.    
  
  
 

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5. 
OBJECTIVES  
5.1 
Primary Objectives  
To determine differences in the release profile of NOFQ from immune cells in the blood 
(neutrophils, eosinophils) between patients with sepsis admitted to the intensive care 
unit and healthy volunteers.  
  
5.2 
Secondary Objectives  
Determine the identity of cells of the immune system containing NOFQ in healthy 
volunteers and patients with sepsis admitted to the intensive care unit, and any 
differences between sepsis and severe sepsis  
Determine NOFQ release in  
Neutrophils  
Eosinophils  
  
Determine differences in expression of N/OFQ and NOP genes in immune cells from 
septic and healthy volunteers using immunofluorescence and PCR as surrogates  
  
  
  
5.3 
Exploratory End Point (where applicable)  
  
  
 

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6. 
STUDY DESIGN  
6.1 
Summary of Trial Design  
Case control study to determine the types of immune cells capable of releasing NOFQ, 
and how this differs in patients diagnosed with sepsis/septic shock, and healthy 
volunteers using a fluorescence microscopy test.  Differences in this release profile will 
be determined in septic patients and healthy volunteers, and correlated with mortality, 
length of hospital and intensive care stay and physiological scores.  
  
Participants will be recruited sequentially; screening, recruitment, sampling and testing 
will occur concurrently.  Patients with sepsis will be identified by discussion with the 
consultant responsible for the intensive care unit.  Healthy volunteers will be 
approached within the Department of Cardiovascular Sciences, University of Leicester.    
  
Following consent or assent, patients will be required to have a single 30ml sample of 
blood taken, within 48 hours of intensive care admission, after which no further active 
participation is required.  They will be followed until hospital discharge or death, and 
these dates recorded, and may withdraw consent at any time.  Their General 
Practitioner will be contacted at 30 days for follow up of mortality, and they will be 
informed of the patient’s participation in the study.  
  
Patient medical records will be accessed to 1) ensure that there is no contraindication 
to blood sampling, 2) they do not fall within the exclusion criteria, 3) apply physiological 
scores to grade severity, 4) ensure that the diagnosis of sepsis is valid.  
  
Healthy volunteers will be required to have a single 30ml sample of blood taken 
following consent, and will be interviewed to ensure that they do not fall within the 
exclusion criteria and that there is no contraindication to blood sampling.  
  
Once collected, all samples will be pseudoanonymised and assigned a case ID, such 
that participants will not be identifiable.  The case ID and original participant details will 
be linked in a sample log, stored securely.  For purposes of research governance, it 
would be possible to disclose the anonymisation, although the requirement for this is 
unlikely for a non-interventional study.  
  
The duration of the study is from January 2016-December 2018.  
  
6.2 
Primary and Secondary Endpoints/Outcome Measures  
To determine differences in the release profile of NOFQ from immune cells in the blood 
between patients with sepsis admitted to the intensive care unit and healthy volunteers  
  
Determine the identity of cells of the immune system containing NOFQ in healthy 
volunteers and patients with sepsis admitted to the intensive care unit, and any 
differences between sepsis and severe sepsis  
Determine NOFQ release in  
Neutrophils  
Eosinophils  
  
   
  
Outcome measures  
  
  
The release of NOFQ will be determined by fluorescence of Chinese Hamster Ovary 
cells, expressing NOP, coupled to Calcium release and stained with a calcium sensitive 

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fluorescent dye.  Relative fluorescence (change / basal) will be measured following 
layering of immune cells, and degranulation.  A relative fluorescence of >1.8 indicates 
a response, and the presence of NOFQ.  Measures will be number of “responder” cells, 
fluorescent change by time, area under the time / fluorescence curve.  
Cell count and differential proportion of immune cell populations  
Physiological scores  
Mortality in-hospital, at 30 days   
Length of ITU stay, and hospital stay  
   
  
 

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7. 
TRIAL PARTICIPANTS  
7.1 
Overall Description of Trial Participants  
Patients admitted to the intensive care unit with a diagnosis of sepsis will be identified 
by discussion with the responsible consultant and nursing staff.  For the purposes of 
this study, patients must have a diagnosis of sepsis by Sepsis-3 criteria; with 
microbiological evidence or suspicion of infection (positive blood culture, urine dipstick, 
compatible history or examination, radiographic evidence)  
  
Patients (or their personal consultee where the patient lacks capacity) will be provided 
with a Participant Information Sheet and consent/assent taken if the participant (or their 
personal consultee) agrees.  As an early blood sample is required, the time to read and 
consider the PIS is limited.  
  
Healthy volunteers will be approached within the Department of Cardiovascular 
Sciences, and provided with the PIS, with consent taken by one of the investigating 
team.  
  
7.2 Inclusion Criteria  
For septic patients;   
1. Participant is willing and able to give informed consent for participation in the study, 
or if lacking capacity, a next of kin or advocate is willing and able to give assent for 
participation in the study.  Must be able to read and understand English.  
2. Male or Female, aged 18 years or above.  
3. Diagnosed with sepsis (see 7.1) and admitted to the intensive care unit.    
4. Able (in the Investigators opinion) and willing to comply with all study requirements.  
5. Willing to allow his or her General Practitioner and consultant, if appropriate, to be 
notified of participation in the study.  
  
Healthy Volunteers;   
1. Participant is willing and able to give informed consent for participation in the study.  
Must be able to read and understand English. 2. Male or Female, aged 18 years or 
above and be   
3. In good health.   
4. Have had no course of medication, whether prescribed or over-the-counter, in the 
four weeks before first study dose and no individual doses in the final two weeks 
other than mild analgesia, vitamins and mineral supplements or, for females, oral 
contraceptives  
5. Able (in the Investigators opinion) and willing to comply with all study requirements.  
6. Willing to allow his or her General Practitioner and consultant, if appropriate, to be 
notified of participation in the study.  
  
  
7.3 
Exclusion Criteria  
1. Conditions which may make phlebotomy hazardous to the participant (such as 
significant bleeding disorders or anaemia, or allergy), or to the investigator (blood 
viral infection).  
2. Any significant disease or disorder which, in the opinion of the Investigator, may 
either put the participants at risk because of participation in the study, or may 
influence the result of the study, or the participant’s ability to participate in the 
study.  
3. Participants who have participated in another research study involving an 
investigational product in the past 12 weeks.  

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8. 
STUDY PROCEDURES  
  
8.1 
Informed Consent  
Following screening and identification (see 8.2), participants or their personal consultee 
(where the participant lacks capacity) will be approached by the investigating team.  
Practically, the consenting procedure and blood sampling should occur within the first 
48 hours of ITU admission.  If required, the participant will be allowed as much time as 
wished up to 48hours post intensive care admission to consider the information, and 
the opportunity to question the Investigator, their GP or other independent parties to 
decide whether they will participate in the study.  Written Informed Consent will then 
be obtained by means of participant dated signature and dated signature of the person 
who presented and obtained the informed consent. The person who obtained the 
consent must be suitably qualified and experienced, and have been authorised to do 
so by the Chief/Principal Investigator as detailed on the Delegation of Authority and 
Signature log for the study. The original signed form will be retained at the study site 
within the Trial Master File (TMF) or Investigator Site File (ISF). A copy of the signed 
Informed Consent will be given to participants and a copy retained in the participant 
medical notes.  
  
For those patients lacking capacity (as defined by the Mental Capacity Act 2005), their 
friend or relative (personal consultee) will be asked to assent for them, as per the 
process above.  If the patient later regains capacity, they will be approached in order 
to confirm consent for the study.  If they withdraw their consent, their data and samples 
will be destroyed.  If the patient dies without regaining capacity, they will be included 
in the trial, and samples retained.  
  
At the time of consent/assent, a Case Report Form (CRF) will be completed, including 
safety information (medications, past medical history, allergies), demographic 
information (age, gender), and study information (lactate, blood pressure, oxygen 
requirement, pulse, and likely source of sepsis).  These data will already exist on 
admission to the intensive care unit.  After consent and provided that there are no 
exclusions (see 7.3), a blood sample (1 x 30ml EDTA) will be collected by the 
investigator, either by venepuncture, or by sampling an indwelling arterial line or central 
venous catheter, using a standard aseptic non touch technique.  
  
  
8.2 
Screening and Eligibility Assessment  
  
Participants will be identified by discussion with the consultant in charge of the 
intensive care unit, nursing staff and screening medical records.  
  
Potential participants (or their personal consultee where participants lack capacity) will 
be approached by the investigating team for the consent/assent procedure.  A 
participant information sheet (see appendix) will be provided, and the opportunity to 
ask questions about the study.  Due to the need to obtain an early sample on Day 1 of 
intensive care admission, provided the participants or advocates agree, they must sign 
the latest approved consent form prior to further study activities.  
  
Demographics  
The date of birth and gender will be recorded.   
  

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Medical History  
Details of illness likely to present a hazard to the participant (significant anaemia, bleeding 
disorder, allergies) or the researcher (transmissible blood viral infections) during blood 
sampling will be recorded.    
The details of the current intensive care admission will be documented on the CRF 
including reason for admission, and medications on admission.  
  
Physical Examination  
 
  
A  
physical examination – or access to the documented observations will be sought to 
confirm the diagnosis of sepsis (7.1 - lactate, blood pressure, oxygen requirement, 
pulse urine output and likely source of sepsis).   
  
Laboratory Tests   
No new laboratory tests will be required for the screening and eligibility phase, although 
tests already completed as part of the routine care for the patient, including   Full Blood 
Count, Urea and Electrolytes, Liver Function Tests, Blood Gas Analysis, CReactive 
Protein and the results of microbiological culture will be accessed in order to determine 
the presence of sepsis (7.1).  
  
Radiology / Imaging Procedures  
No new radiology or imaging procedures will be undertaken for the purposes of this 
study.  If radiological evidence of infection (such as a chest infection or pneumonia) 
exists, this will be screened to determine eligibility and the presence of sepsis (as 
defined in 7.1).  
  
  
8.3 
Baseline Assessments  
Baseline assessment will include severity of assessment, measured using existing 
patient data collected as part of routine care.  The purpose of this assessment is to 
determine the presence of sepsis/septic shock.  
- 
Lactate  
- 
Blood Pressure, Pulse  
- 
Arterial blood gas  
- 
Blood glucose  
- 
Microbiological samples (blood, urine, sputum, fluid for culture) -  
Presence 
of vasoactive medications and inotropes  
  
  
8.4 
Randomisation and Codebreaking (if applicable)  
This study will be conducted as a case control protocol and is non-interventional.  
Samples will be pseudoanonymised once collected; the code for the Case ID will be 
stored securely to enable codebreaking for governance purposes.  
Subject numbers will be assigned sequentially as each subject enters the study.    
  
8.5 
Subsequent Assessments  
Patients will have blood sampled (as per the protocol) within 48 hours of their intensive 
care admission.  If a patient is readmitted to the intensive care unit with another 
diagnosis of sepsis after previous enrolment, they will not be re-enrolled and will be 
excluded from further participation.  
The General Practitioner for enrolled patients will be contacted to inform them of their 
participation.  Healthy volunteers will have a single sample of blood taken.    
  

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8.6 
Definition of End of Trial  
The end of trial is when the mortality follow-up is complete from the last recruited 
patient.    
  
8.7 
Discontinuation/Withdrawal of Participants from Study  
 Each participant has the right to withdraw from the study at any time.  In addition,  the 
investigator may discontinue a participant from the study at any time if the  investigator 
considers it necessary for any reason including:   
Ineligibility (either arising during the study or retrospective having been overlooked at 
screening)  
Significant protocol deviation  
Consent withdrawn Lost 
to follow up  
The reason for withdrawal will be recorded in the CRF.    
If the participant is withdrawn due to an adverse event, the investigator will arrange for 
follow-up visits or telephone calls until the adverse event has resolved or stabilised.    
  
8.8 
Source Data  
Source documents are original documents, data, and records from which participants’ 
CRF data are obtained.  These include, but are not limited to, hospital records (from 
which medical history and previous and concurrent medication may be summarised 
into the CRF), clinical and office charts, laboratory and pharmacy records, diaries, 
microfiches, radiographs, and correspondence.  
CRF entries will be considered source data if the CRF is the site of the original 
recording (e.g., there is no other written or electronic record of data All documents will 
be stored safely in confidential conditions.  On all study-specific documents, other than 
the signed consent, the participant will be referred to by the study participant 
number/code, not by name.  
  
  
 

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9. 
TREATMENT OF TRIAL PARTICIPANTS  
9.1 
Description of Study Treatment   
The trial is non-interventional.  Aside from blood sampling (as described in the protocol 
above), there will be no drugs or interventions administered.  
  
9.2 
Storage of Study Equipment or Related apparatus  
The study requires phlebotomy equipment with no special storage requirements.  
Blood samples will be stored at 18-22 C following sampling.   Storage for use in 
related studies will be at -20 C.  Samples for PCR will be stored at -20◦C.  
  
9.3 
Compliance with Study Treatment  
As this is a non-interventional trial, there will be no measures of medication or 
intervention compliance.  
If patients become ineligible, their data will be retained but any samples will be 
destroyed.  If a patient withdraws consent prior to the completion of their involvement, 
their data and samples will be destroyed.  
  
 

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10. 
SAFETY REPORTING  
10.1 Definitions  
  
10.1.1 Adverse Event (AE)  
An AE or adverse experience is:  
Any untoward medical occurrence in a patient or clinical investigation participants, 
which does not necessarily have to have a causal relationship with this treatment. An 
AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the study, whether 
or not considered related to the study.  
  
10.1.3 Severe Adverse Events  
To ensure no confusion or misunderstanding of the difference between the terms 
"serious" and "severe", which are not synonymous, the following note of clarification 
is provided:  
The term "severe" is often used to describe the intensity (severity) of a specific event 
(as in mild, moderate, or severe myocardial infarction); the event itself, however, may 
be of relatively minor medical significance (such as severe headache).  This is not the 
same as "serious," which is based on patient/event outcome or action criteria usually 
associated with events that pose a threat to a participant's life or functioning.  
Seriousness (not severity) serves as a guide for defining regulatory reporting 
obligations.  
  
10.2 Reporting Procedures for All Adverse Events  
As this is a non-interventional study, AE’s and SAE’s are unlikely, and therefore 
AS/SAE reporting is not applicable to this type of study.  
  
  
  
11. 
STATISTICS  
  
11.1 Description of Statistical Methods  
Fluorescence regions of interest, defined as all CHO cells adjacent to and surrounding 
the immunocyte of interest will be marked by the researcher.  Basal fluorescence – the 
mean fluorescence determined per cell (region of interest) for the first 10 seconds of 
imaging.  Relative fluorescence (change in fluorescence / basal) will be determined for 
each cell, at each time point.  
Cells expressing a relative fluorescence of 1.8 at any point will be classed  
“responders”; other CHO cells adjacent to the immune cell, and with relative 
fluorescence <1.8 will be classed as nonresponders.   The proportion of responders 
will be compared using 
2 with Fisher’s exact test between healthy volunteers and 
intensive care patients.  
Presence of nociceptin peptide, receptor, and identifying immune cell markers 
will be assessed qualitatively by fluorescence microscopy.  
Presence of nociceptin peptide, and receptor encoding DNA will be assessed 
using quantitative PCR with comparison of CT values between groups.  
  
  
11.2 The Number of Participants  
This is a pilot study to develop this assay for use in comparing patient groups, and will 
be used to develop further understanding of the discriminative power of this assay.  

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Therefore, it is not possible to give a definitive number of participants.  8 participants 
per group is deemed practical and realistic.  
  
11.3 The Level of Statistical Significance  
  
A conventional level of statistical significance of p<0.05 will be used.  
  
11.4 Criteria for the Termination of the Trial.  
The trial will be terminated when the number of participants is >5 per group and the 
nociceptin release profile has been determined for septic and healthy volunteers.  
  
11.5 Procedure for Accounting for Missing, Unused, and Spurious Data.  
Where data is missing and cannot be reconciled, the case to which it related will be 
excluded from the analysis.  All data will be retained, and through the ethics process, 
permission is sought to reuse this data in subsequent studies.  The significance of the 
nociceptin release profile in sepsis is unknown, and data collected via the CRF for the 
purposes of this study may give additional information in relation to this.  
  
11.6 Procedures for Reporting any Deviation(s) from the Original Statistical Plan  
Depending on the sensitivity of the test, or the characteristics of the fluorescence 
observed, it may be necessary to examine other measures.  Where necessary, this will 
be reported in any written report, and will be submitted as a protocol amendment.  
  
11.7 Inclusion in Analysis  
All participants giving consent for venepuncture will be included in the data analysis.  
For those patients who lacked capacity to consent, and for whom assent from next of 
kin was gained, if permission was withdrawn when they regained capacity, samples 
and data would be destroyed.  
  
  
12. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS  
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.  
  
  
 

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13. 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES  
The study will be conducted in accordance with the current approved protocol, ICH 
GCP, relevant regulations and standard operating procedures.   
  
The University of Leicester as sponsor operate a risk based audit programme to which 
this study will be subject.  
  
 

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14. 
CODES OF PRACTICE AND REGULATIONS  
14.1 Ethics  
Describe ethical considerations relating to the trial. Include general and study specific 
ethics considerations.  
  
14.2 Sponsor Standard Operating Procedures  
All relevant Sponsor SOPs will be followed to ensure that this study complies with all 
relevant legislation and guidelines   
  
14.3 Declaration of Helsinki  
The Investigator will ensure that this study is conducted in full conformity with the 
current revision of the Declaration of Helsinki (last amended October 2000, with 
additional footnotes added 2002 and 2004).  
  
14.4 ICH Guidelines for Good Clinical Practice  
The Investigator will ensure that this study is conducted in full conformity with relevant 
regulations and with the ICH Guidelines for Good Clinical Practice (CPMP/ICH/135/95) 
July 1996.  
  
14.5 Approvals   
Once Sponsor authorisation has been confirmed, the protocol, informed consent form, 
participant information sheet and any proposed advertising material will be submitted 
to an appropriate Research Ethics Committee (REC), regulatory authorities (MHRA in 
the UK), and host institution(s) for written approval.    
Once Sponsor authorisation has been confirmed, the Investigator will submit and, 
where necessary, obtain approval from the above parties for all substantial 
amendments to the original approved documents.     
  
14.6 Participant Confidentiality  
The trial staff will ensure that the participants’ anonymity is maintained.  The 
participants will be identified only by initials and a participants ID number on the CRF 
and any electronic database.  All documents will be stored securely and only accessible 
by trial staff and authorised personnel. The study will comply with the Data Protection 
Act which requires data to be anonymised as soon as it is practical to do so.    
  
14.7 Other Ethical Considerations  
This study will require the recruitment of patients admitted to the intensive care unit.  
These patients may lack capacity; due to the requirement for a blood sample on the 
first day of admission, assent may be obtained from the patient’s friend or relative, their 
personal consultee.  Should the patient later withdraw their consent if they regain 
capacity, their sample and any data will be destroyed.  
Samples will be collected from staff and students of the Department of Cardiovascular 
sciences.  All participants will be free to withdraw their consent at any point in the study.  
  
  
15. 
DATA HANDLING AND RECORD KEEPING  
All study data will be entered on a Microsoft Excel Spreadsheet.  Data analysis will be 
carried out in the Statistical Package for the Social Sciences (SPSS).    
The participants will be identified by a study specific participants number and/or code 
in any database.  The name and any other identifying detail will NOT be included in 
any study data electronic file.   
  

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Data will be stored in pseudoanonymised form in the central filestore at the University 
of Leicester.  The imaging data will be stored on the Research File Store at the 
University of Leicester.    
  
The Case ID code will be stored securely on the sampling log in paper form in the ISF.  
  
  
 

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16. 
STUDY GOVERNANCE  
16.1 Trial Steering Committee (TSC)  
 
   
N/A  
  
  
16.2  Data Safety Monitoring Committee (DSMC)  
 
   
N/A  
  
  
 

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17. 
FINANCING AND INSURANCE  
This study is supported by grants from the BBSRC (approved) which covered the 
development and validation of the bioassay, and the use of samples from healthy 
volunteers.  A further grant is pending from the Royal College of Anaesthetists to cover 
the application of this bioassay in septic patients.   
  
Research costs - University  
Description  Lab consumables – Tissue culture (6 months)  
Amount  
£300  
Description  Lab consumables – Fluorescence  
Amount  
£188.24  
Description  Lab consumables – Cell separation  
Amount  
£1492.00  
Description  Use of Advanced Imaging Facility and  
Microscope  
Amount  
£400  
Description  Consumables – Blood sampling  
Amount  
£42.39  
  
NHS Treatment Costs  
As this is a non interventional study, there are no NHS treatment costs  
  
NHS Support Costs  
As this is a non interventional study, the blood sampling is to be performed by the 
investigator under an honorary contract with the University hospitals of Leicester, there 
are no NHS Support costs.    
  
  
  
18. 
PUBLICATION POLICY  
Data collected as part of this study will be disseminated and published in appropriate 
scientific fora, including meetings, conferences, in journal publications on- and offline.  
Images taken using microscopy will be published as part of this.  However, no participant 
identifiable information will be published as part of dissemination.  
  
  
 

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19. 
REFERENCES  
References  
Arjomand, J., Cole, S., & Evans, C. J. (2002). Novel orphanin FQ/nociceptin transcripts are expressed 
in human immune cells. Journal of Neuroimmunology, 130(1-2), 100-108.   
Dellinger, R. P., Levy, M. M., Rhodes, A., Annane, D., Gerlach, H., Opal, S. M., . . . Surviving 
Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. (2013). Surviving 
sepsis campaign: International guidelines for management of severe sepsis and septic shock:  
2012. Critical Care Medicine, 41(2), 580-637. doi:10.1097/CCM.0b013e31827e83af [doi]  
Fiset, M. E., Gilbert, C., Poubelle, P. E., & Pouliot, M. (2003). Human neutrophils as a source of 
nociceptin: A novel link between pain and inflammation. Biochemistry, 42(35), 10498-10505.   
Kumar, A., Roberts, D., Wood, K. E., Light, B., Parrillo, J. E., Sharma, S., . . . Cheang, M. (2006). 
Duration of hypotension before initiation of effective antimicrobial therapy is the critical 
determinant of survival in human septic shock. Critical Care Medicine, 34(6), 1589-1596. 
doi:10.1097/01.CCM.0000217961.75225.E9 [doi]  
Lee, H., & Jeon, H. (2013). Plasma concentrations of nociceptin/orphanin FQ: Comparison of levels 
after general and neuraxial anesthesia for arthroscopic knee surgery. Korean Journal of 
Anesthesiology, 65(4), 327-330.   
Meunier, J. C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., . . . Monsarrat, B. 
(1995). Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 
receptor. Nature, 377(6549), 532-535. doi:10.1038/377532a0 [doi]  
Nothacker, H. P., Reinscheid, R. K., Mansour, A., Henningsen, R. A., Ardati, A., Monsma, F. J.,Jr, .  
. . Civelli, O. (1996). Primary structure and tissue distribution of the orphanin FQ precursor. 
Proceedings of the National Academy of Sciences of the United States of America, 93(16),  
8677-8682.   

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Peluso, J., LaForge, K. S., Matthes, H. W., Kreek, M. J., Kieffer, B. L., & Gavériaux-Ruff, C. (1998). 
Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system 
and immune cells. Journal of Neuroimmunology, 81(1), 184-192.   
Peluso, J., Gaveriaux-Ruff, C., Matthes, H. W., Filliol, D., & Kieffer, B. L. (2001). Orphanin 
FQ/nociceptin binds to functionally coupled ORL1 receptors on human immune cell lines and 
alters peripheral blood mononuclear cell proliferation. Brain Research Bulletin, 54(6), 655-660.  
doi:S0361923001004828 [pii]  
Reinscheid, R. K., Nothacker, H. P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., . . . 
Civelli, O. (1995). Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled 
receptor. Science (New York, N.Y.), 270(5237), 792-794.   
Stamer, U. M., Book, M., Comos, C., Zhang, L., Nauck, F., & Stuber, F. (2011). Expression of the 
nociceptin precursor and nociceptin receptor is modulated in cancer and septic patients.  
British Journal of Anaesthesia, 106(4), 566-572.   
Thompson JP, Serrano-Gomez A, McDonald J, Ladak N, Bowrey S, & Lambert DG. (2013). The 
nociceptin/orphanin FQ system is modulated in patients admitted to ICU with sepsis and after 
cardiopulmonary bypass. PLoS ONE [Electronic Resource], 8(10), e76682. 
doi:http://dx.doi.org/10.1371/journal.pone.0076682  
Wang, J. B., Johnson, P. S., Imai, Y., Persico, A. M., Ozenberger, B. A., Eppler, C. M., & Uhl, G. R.  
(1994). cDNA cloning of an orphan opiate receptor gene family member and its splice variant.  
FEBS Letters, 348(1), 75-79.   
Wick, M. J., Minnerath, S. R., Roy, S., Ramakrishnan, S., & Loh, H. H. (1996). Differential 
expression of opioid receptor genes in human lymphoid cell lines and peripheral blood 
lymphocytes. Journal of Neuroimmunology, 64(1), 29-36.   

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Williams, J. P., & Thompson, J. P. (2008). Nociceptin and urotensin-II concentrations in critically ill 
patients with sepsisâ€ . British Journal of Anaesthesia, 100(6), 810-814. 
doi:10.1093/bja/aen093  
Williams, J. P., Thompson, J. P., McDonald, J., Barnes, T. A., Cote, T., Rowbotham, D. J., & 
Lambert, D. G. (2007). Human peripheral blood mononuclear cells express 
nociceptin/orphanin FQ, but not mu, delta, or kappa opioid receptors. Anesthesia and  
Analgesia, 105(4), 998-1005, table of contents. doi:105/4/998 [pii]  
   
 

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20. 
APPENDIX A: STUDY FLOWCHART  
 

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21. 
APPENDIX B: SCHEDULE OF PROCEDURES