Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient
Conditions: Asthma, COPD, Heart Failure, Community-acquired Pneumonia, Healthy, Breathlessness
Sponsor: University of Leicester

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
 
 
Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient 
Short Title: EMBER Study 
Sponsor Reference No: 
UNOLE 0569 
Ethics Ref:  
 
     
     IRAS 198921 
 
Date and Version No: 
1 April 2018 : Version 4  
Chief Investigator: 
Professor Salman Siddiqui 
Investigators:  
Professor Mike Barer, Dr Caroline Beardsmore, Professor Chris 
Brightling, Professor Tim Coats, Dr Rebecca Cordell, Dr Martha Clokie, 
Dr Rachael Evans, Dr Robert Free, Dr Erol Gaillard, Professor Alison 
Goodall, Dr Neil Greening, Dr Ian Loke, Dr John Le Quesne, Professor 
Paul Monks, Professor Rachel Munton, Professor Leong Ng, Dr Hitesh 
Pandya, Dr James Reynolds, Dr Damian Roland, Dr Matthew 
Richardson, Dr Kimuli Ryanna, Sir Nilesh Samani, Professor Jaqui Shaw, 
Professor Ian Squire, Dr Adrian Stanley, Professor Michael Steiner, 
Professor Toru Suzuki, Professor Paul Thomas, Professor Andrew 
Wardlaw, Dr Geritt Woltmann. 
 
 
Sponsor:  
University of Leicester : Reference Number 0569 
Funder: 
Medical Research Council/Engineering and Physical Sciences Research 
Council  UK, Midlands Asthma and Allergy Research Association 
(MAARA) 
 
 
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. 
 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
Authors 
 
 
 
Professor Salman Siddiqui, Professor of Airways Disease, University of Leicester/University 
Hospitals of Leicester. 
 
Dr Neil Greening, NIHR post doctoral fellow, Associate Professor, University of Leicester/ University 
Hospitals of Leicester. 
 
Dr Hitesh Pandya, Clinical Senior Lecturer in Paediatric Airways Disease, University of Leicester/ 
University Hospitals of Leicester. 
 
Dr Erol Gaillard, Clinical Senior Lecturer, University of Leicester/ University Hospitals of Leicester 
 
Dr Caroline Beardsmore, Senior Lecturer in Paediatric Airways Disease, University of Leicester/ 
University Hospitals of Leicester. 
 
Professor Tim Coats, Professor of Emergency Medicine, University of Leicester/ University 
Hospitals of Leicester. 
 
Dr Matthew Richardson, Post-Doctoral Statistician, University of Leicester. 
 
Dr Rob Free, Post-Doctoral Information, computing and technology lead, University of Leicester. 
 
Professor Paul Monks, Professor of Analytical Chemistry, University of Leicester. 
 
Dr Rebecca Cordell, Post-doctoral researcher analytical chemistry, University of Leicester. 
 
Professor Paul Thomas, Professor of Analytic Chemistry, Loughborough University. 
 
Dr Wadah Ibrahim, Clinical Research Fellow, University of Leicester/University Hospitals of 
Leicester 
 
Amisha Singapuri, Clinical Trial Project Manager, University of Leicester. 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
Signature Page 
 
Chief Investigator Name: 
Prof Salman Siddiqui 
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) 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
TABLE OF CONTENTS 
1. 
AMENDMENT HISTORY 
6 
2. 
SYNOPSIS 
8 
3. 
ABBREVIATIONS 
11 
4. 
BACKGROUND AND RATIONALE 
 13 
5. 
OBJECTIVES 
14 
5.1 
Primary Objectives 
14 
5.2 
Secondary Objectives 
15 
5.3 
Exploratory End Point (where applicable) 
15 
6. 
STUDY DESIGN 
 16 
6.1 
Summary of Trial Design 
16 
6.2 
Summary of additional assessment 
 18 
7. 
TRIAL PARTICIPANTS 
 20 
7.1 
Overall Description of Trial Participants 
20 
7.2 
Inclusion Criteria 
 20 
7.3 
Exclusion Criteria  
20 
8. 
STUDY PROCEDURES 
21 
8.1 
Informed Consent 
21 
8.2 
Screening and Eligibility Assessment 
22 
8.3 
Baseline Assessments 
23 
8.4 
Breath sampling procedures   
  
 
8.5 
Subsequent Assessments 
 
8.6  
Definition of End of Study 
30 
8.7 
Source Data 
30 
9. 
SAFETY REPORTING 
30 
9.1 
Definitions 
30 
9.2 
Reporting Procedures for All Adverse Events 
32 
9.3 
Reporting Procedures for Serious Adverse Events 
32 
10. STATISTICS 
32 
10.1 Description of Statistical Methods 
32 
10.2 The Number of Participants 
34 
10.3 The Level of Statistical Significance 
34 
10.4 Criteria for the Termination of the Trial 
34 
10.5 Procedure for Accounting for Missing, Unused, and Spurious Data 
34 
10.6 Procedures for Reporting any Deviation(s) from the Original Statistical Plan 
34 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
10.7 Inclusion in Analysis 
34 
11. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
34 
12. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
34 
13. CODES OF PRACTICE AND REGULATIONS 
35 
14. DATA HANDLING AND RECORD KEEPING 
36 
14.1 Ethics 
 
14.2 Sponsor Standard Operating Procedures 
 
14.3 Declaration of Helsinki 
 
14.4 ICH Guidelines for Good Clinical Practice 
 
14.5 Approvals 
 
14.6 Participant Confidentiality 
 
14.7    Other Ethical Considerations 
 
15. STUDY GOVERNANCE 
37 
15.1 Clinical Steering Committee (TSC) 
37 
15.2 Data Safety Monitoring Committee (DSMC) 
38 
16. FINANCING AND INSURANCE 
38 
17. PUBLICATION POLICY 
39 
18. REFERENCES 
39 
19. APPENDIX A:  RECIVA BREATH SAMPLING DEVICE 
41 
20. APPENDIX B: RECIVA CE MARKING AUTHORISATION 
42 
21. APPENDIX C: Interim analysis and sample size calculation                                                                              45 
         21.1 EMBER Sample Size Estimates                  
 
 
 
 
 
 
 45 
         21.2 Closed formula for logistics regression models  
 
 
 
 
 
 45 
         21.3 Closed formula for sensitivity and specificity of disease biomarkers 
 
 
 
 46 
         21.4 Summary 
 
 
 
 
 
 
 
 
 
 
 46 
         21.5 Recruitment plan 
 
 
 
 
 
 
 
 
  
 47 
         21.6 Paediatric recruitment plan: acute study 
 
 
 
 
 
               47 
         21.7 Stable disease recruitment plan                           
 
 
 
 
 
 47 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
 
1. AMENDMENT HISTORY 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Author(s) of changes 
Details of Changes made 
 
3 
1/11/17 
Dr Neil Greening 
Prof Salman Siddiqui 
Dr Wadah Ibrahim 
 
SA#02 
4 
1/04/18 
Dr Wadah Ibrahim 
Amisha Singapuri 
Clarifications, Typos, Additional Author. 
- 
Follow up duration extended to 6 
months from 8-16 weeks.  
- 
Inclusion Criteria (i) amended to say 
that all patients need to be able to 
give consent not just the acute 
patients 
- 
The number of visits the Healthy 
Volunteers will have is 2 instead of 
3.  
- 
ACQ questionnaire in the table 
schedule was supposed to have 
been the AQLQ questionnaire, 
however this typo was missed at the 
last amendment. ACQ amended to 
AQLQ 
- 
Added the NASA Task Load index.  
- 
CRP & FBC blood tests have been 
removed from the pneumonia V1b 
visit.  
- 
Quadriceps Ultrasound removed in 
Heart failure patients.  
- 
4 Meter Gait in Heart failure 
subjects removed from Visit V1a and 
added to. This test has been 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
completely removed from Healthy 
Volunteers. 
- 
Physical exam removed from Visit 2 
in Healthy Volunteers. 
- 
Review of AE & SAE at Visit 1a 
removed for Paediatric patients. 
- 
Lung function clarification given that 
spirometry is both Pre and Post 
bronchodilator  to match the patient 
information sheets. 
- 
Removed the questionnaire from 
the appendix of the protocol. 
 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
2. SYNOPSIS 
 
Study Title 
Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. 
Internal ref. no. 
Sponsor Reference 0569 
Trial Design 
Prospective Acute Care ‘Real Life’ Observational Study  
Trial Participants 
Patients with acute breathlessness in the following (post-acute pathway 
confirmed) strata:  
 
(i) Acute Heart failure, characterised by a clinician diagnosis, and a proto-typical 
response to treatment.  
 
(ii) Community acquired pneumonia, defined as new radiological consolidation 
responsive to treatment according to the British Thoracic Society (BTS) 
community acquired pneumonia guidelines. 
 
(iii) Adult exacerbations of asthma and COPD, defined as a clinician diagnosis 
with objective confirmatory evidence of disease based upon historical or 
prospective measurements, requiring an acute change in treatment (e.g. 
antibiotics, oral corticosteroids or increased bronchodilator therapy) 
 
(iv) Paediatric exacerbations of acute childhood wheeze and or confirmed 
asthma  
 
(v) Age and/or Home environment matched Adult/Sibling/Parent/Spousal 
healthy  volunteers, who have no prior history of asthma, COPD, heart failure 
and have not been admitted to hospital with community acquired pneumonia 
within 6 weeks of the baseline study visit. Additonal healthy volunteers will also 
be recruited from local databses. 
 
(vi) Stable state aged matched participants with a confirmed  diagnosis  of  
asthma, COPD or heart failure , that have not had and exacerbation of their 
disease within the preceding 6 weeks.  
Planned Sample Size 
An interim analysis was conducted in September 2017 to determine the sample 
size of the ember acute study. Guided by literature search, a panel of 10 pre 
specified aldehyde biomarkers derived from GCMS analysis were supplied. The 
biomarkers were normalised to a common standard and were not background 
subtracted. 
A closed formula from Hsieh et al, Statis med 1998, was utilised to calculate 
sample sizes from logistic regression models of the 10 aldehydes with acute 
breathlessness as the outcome measure. The sample size estimates are also 
relevant to acute class comparisons vs. the sum of other acute classes. 
Based upon the samples size estimates we would have an 80% power at the 5 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
% significance level to detect an odds ratio of association of 1.2 for a given 
disease class with minimum of 55 patients per disease class. 
 
Discovery Phase (years 1-2): Acute Heart failure (n=55-100), Community 
acquired pneumonia (n=55-100),  adult exacerbations of 
asthma and COPD (n=110-200), paediatric exacerbations of acute 
childhoodwheeze and/or confirmed asthma (n=50-100), 
Age/Home environment matched Adult/Sibling/Parent/Spousal healthy 
volunteers (n=55-150 adults/50 children) 
 
Replication Phase (years 3-4): Acute Heart failure (n=55-100),Community 
acquired pneumonia (n=55-100),  adult exacerbations of asthma and COPD 
(n=110-200), paediatric exacerbations of acute childhood 
wheeze and/or confirmed asthma  (n=25-50), Age/Home 
environment matched Adult/Sibling/Parent/Spousal healthy 
volunteers (n=55-75 adult/25 children) 
 
Additional patients with stable COPD/Asthma/Heart failure  n=200 
Total Sample Size = 550-1250 patients. 
Follow-up duration 
Acute care participants will have up to 2 visits whilst an inpatient followed by a 
‘Post-Acute Event Recovery’ at a minimum of 6 weeks post discharge and up to 
6 months. 
Stable state participants and healthy volunteers will have 2 visits over 16 
weeks. 
All participants will have outcome data 2 years post recruitment. 
Planned Trial Period 
3 years 
Primary Objective 
To evaluate the sensitivity, specificity, positive and negative predictive value of 
metabolomic biomarkers in exhaled breath samples to identify acute 
breathlessness,  defined as one or more of (i) patient defined acute 
breathlessness and/or a (ii) 1 unit increase in Extended Medical Research 
Council breathlessness score (eMRC). 
Secondary Objectives  
1) To replicate selected metabolomic breath biomarker panels of acute 
breathlessness using a combination of both (i) discovery technologies 
(including GC-MS) and (ii) point of care auto tuned breath sensing 
devices (including linear IMS, CMS, PTR-MS).  
 
2) To evaluate the primary endpoint stratified by breathlessness as 
follows (i) change in eMRC by 1 unit, (ii) patient defined breathlessness 
and (iii) 100 mm visual analogue score for breathlessness 
 
To discover and replicate metabolomic breath biomarkers that differentiate (i) 
Acute Heart failure, (ii) Community acquired pneumonia, (iii) Adult 
exacerbations of asthma and COPD 

 
 
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To quantify the level of clinical uncertainty in the primary diagnosis using a 100 
mm visual analogue scale and independent clinical adjudication of case notes 
blinded to the following blood biomarkers (i) CRP (ii) blood eosinophils (iii) BNP 
(iv) Troponin-I but not clinical history and chest X-ray.  
 
To identify  and discover metabolomic breath biomarkers of acute childhood 
wheeze and or confimed asthma. 
To clinically validate metabolomic breath biomarkers generated above using a 
combination of existing pathology markers e.g. BNP, CRP, blood eosinophils 
and troponin-I and other clinical relevant prognostic scoring tools e.g DECAF, 
CURB65 and Heart failure risk calculator http://www.heartfailurerisk.org/[19]  
Exploratory Objectives 
1) To evaluate the dynamic profile of breath biomarkers during (i) the 
acute state, (ii) in the recovery state post exacerbation. 
 
2) To evaluate the relationship between breath metabolomic biomarkers 
and health status/ functional measures: 
- 
Health status measures 
- 
Physical performance 
 
3)  To evaluate the relationship between metabolomic breath biomarkers 
and clinical outcomes including (i) hospital readmission at time points 
up to 2 years (ii) response to standard clinical therapy and (iii) death. 
 
4) To explore and develop breath metabolomic biomarkers of multi-
morbidity 
 
5) To evaluate the relationship between diet, lifestyle and environment 
upon breath metabolomic biomarkers.   
 
6) To generate a biobank of DNA, RNA, urine, plasma, serum and sputum 
samples for future breathomic and other ‘omic molecular pathology 
studies (including proteomic, transcriptomic, genomic and 
metabalomic studies). 
Investigational Medicinal 
Products 
None: Patients will follow their standard acute care pathways 
 
 

 
 
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3. ABBREVIATIONS 
AE 
Adverse event 
AR  
Adverse reaction 
BNP 
B-type natiuretic peptide 
BRC 
Biomedical Research Centre 
BRC 
Biomedical Research Centre 
BTS 
British Thoracic Society 
CAU 
Childrens Assessment Unit 
CDU 
Clinical Decisions Unit 
CI 
Chief Investigator 
CMS 
Compact Mass Spectrometer 
COPD 
Chronic Obstructive Pulmonary Disease 
CRF 
 
Case Report Form 
CRP 
 
C-Reactive Protein 
CTA 
 
Clinical Trials Authorisation 
DDU 
 
Diagnostics Development Unit 
DMS 
 
Differential Mobility Spectrometry 
DSMC 
 
Data Safety Monitoring Committee 
eMRC 
 
Extended Medical Research Council Score 
e-CRF 
 
Electronic Case Report Form 
ECG 
 
Electrocardiogram 
EC 
 
Ethics Committee (see REC) 
ECHO 
 
Echocardiography 
EPSRC 
 
Engineering and Physical Sciences Research Council  
FEV1 
 
Forced Expiratory Volume in 1 second 
FeNO 
 
Fraction Exhaled Nitric Oxide 
GC-IMS 
 
Gas Chromatography Mobility Spectrometry 
GC-FAIMS  
Gas Chromatography Field Asymmetric Mobility Spectrometry 
GC-MS                        Gas Chromatography and Mass Spectroscopy 
GCP 
Good Clinical Practice 
GP 
General Practitioner 
GTAC 
Gene Therapy Advisory Committee 
ICF 
Informed Consent Form 
ICH 
International Conference of Harmonisation 
ISF 
Investigator Site File 
IMP 
Investigational Medicinal Product 
LV 
Left Ventricular 
MAARA 
Midlands Asthma and Allergy Association 

 
 
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MHRA 
Medicines and Healthcare products Regulatory Agency 
MRC 
Medical Research Council 
MRC-EMBER 
MRC East Midlands Breathomics Molecular Pathway Node 
NHS 
National Health Service 
NIHR 
National Institute for Health Reseach 
NRES 
National Research Ethics Service  
PI 
Principal Investigator 
PIL/S 
Participant/ Patient Information Leaflet/Sheet 
PTRMS 
Proton transfer mass spectroscopy device  
PTR-ToF-MS 
Real time breath monitoring using protocon transfer reaction time of 
flight mass spectrometry 
R&D 
NHS Trust R&D Department 
REC 
Research Ethics Committee 
RV 
Right Ventricular 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TMF 
Trial Master File 
UHL 
University Hospitals of Leicester NHS Trust 
VAS 
Visual Analogue Score 
VOCs 
Volatile Organic Compounds 
 
 
 
 
 

 
 
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4. BACKGROUND AND RATIONALE 
 
Patients presenting with acute undifferentiated breathlessness are commonly encountered in 
admissions units & emergency departments across the United Kingdom. The primary mechanisms that 
underpin acute breathless are driven by diseases within the cardio respiratory systems. Cardiorespiratory 
disease accounts for approximately 70 % of acute hospital admissions and therefore is a major health 
priority in the National Health Service (NHS). 
 
The differential diagnosis of undifferentiated breathlessness includes heart failure, community acquired 
pneumonia, exacerbations of chronic obstructive pulmonary disease (COPD), and asthma.  The diagnosis of 
these common conditions is based on a combination of clinical history and emergency diagnostics. 
Importantly, acutely ill patients with multi-morbidity can present and accurate diagnosis remains 
challenging. It is well recognised that when clinicians remain uncertain of the cause of breathlessness, 
patients have a longer hospital stay as well as increased morbidity and mortality [1]. 
 
Currently, blood biomarkers together with clinical, physiological and imaging parameters are used to 
stratify patients with acute breathlessness. Common examples of blood pathology markers utilised in 
routine clinical practice include brain derived natriuretic peptide [BNP] in heart failure [2,3], C reactive 
protein [CRP] and serum procalcitonin in infective bronchitis and pneumonia[4] and blood eosinophils in a 
proportion of exacerbations of asthma and COPD [5].  
 
These blood biomarkers have clinical utility primarily in patients with single pathologies, but have poor 
discriminatory power in patients with multi factorial presentations of acute breathlessness.   For example 
the American College of Emergency Physician recommends addition of a single BNP measurement to 
improve the diagnostic accuracy compared with standard clinical judgment alone in acutely breathless 
patients. BNP levels of <100 and >500 ng/ml can reliably 'rule out' and 'rule in' acute heart failure 
respectively [2,3], but there remain a large number of patients with intermediate values between 100-500 
where there is often significant diagnostic uncertainty.  
 
It is therefore likely that a variety of clinical, radiological features and biomarkers are required to accurately 
identify the cause of acute breathlessness. For example the addition of CRP to conventional clinical rule 
based algorithms modestly improved the positive likelihood ratio of identifying community acquired 
pneumonia at a low, intermediate or high risk in a large european population study [6]. 
 
Therefore better biomarkers are required to identify the cause of breathlessness in acutely unwell patients. 
Ideally these biomarkers would have the following properties (i) they would originate from the target organ 
of interest, (ii) they would significantly add value to conventional risk scoring and diagnostic algorithms in 
acute breathlessness e.g. the DECAF score in COPD[7] exacerbations or the CURB-65 scoring tool in 
community acquired pneumonia[8] ,  (iii) they would be minimally invasive and suitable for rapid point of 
care diagnosis in emergency rooms and on acute admissions units and (iv) they would be have diagnostic 
value in patients with multi factorial acute breathlessness. 
 
 
Breath Analysis and Disease 
 
Exhaled breath analysis of volatile chemicals offers the potential to develop more effective biomarkers in 
acutely breathless patients. The use of breath analysis for disease diagnostics dates back to ancient Greeks 
where physicians used exhaled breath to diagnose different diseases. Breath odours allow correct 
associations to certain diseases. For example, the sweet smell of diabetic ketoacidosis, the fishy smell of 

 
 
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breath associated to liver illness, the urine-like odour of kidney disease and the smell of the breath of 
patients with lung abscesses, caused by the proliferation of anaerobic bacteria [9-12]. 
 
More recently, exhaled breath analysis has demonstrated early proof of concept in the diagnosis of acute 
heart failure [13], ventilator associated pneumonia [14] and stable state airways disease [15]. The validity of 
breath analysis has also been demonstrated in breathless children [16]. This population is likely to prefer 
breath-based tests, as these are minimally invasive. Importantly, a variety of point of care sensors are now 
available to evaluate potential exhaled breath biomarkers in emergency care settings. 
 
The proposed program of research will identify and evaluate the diagnostic and prognostic value of exhaled 
breath metabolomic biomarkers in acute cardio respiratory breathlessness and  common indicator 
diagnoses associated with acute cardio respiratory breathlessness. Specific indicator conditions have been 
selected according to their (i) relatively high prevalence, (ii) high unmet need, mortality and morbidity and 
(iii) need to develop better diagnostic and prognostic algorithms in acute care pathways. 
 
The focus of the research program will be to phenotype using a combination of ‘discovery’ and 
near-patient care breath sampling technologies in acutely breathless patients attending two acute 
admissions units within Leicestershire. The indicator diagnoses of interest are (i) acute heart 
failure, (ii) exacerbations of adult asthma and COPD, (iii) community acquired 
pneumonia and (iv) paediatric exacerbations of acute childhood wheeze and confirmed asthma. Other 
disorders that are associated with acute breathlessness including exacerbations and acute presentations of 
pulmonary fibrosis, acute pulmonary embolism will not be studied within the program due to low 
prevalence, lack of standardised definitions and likely small sample size (pulmonary fibrosis) and adequate 
acute companion diagnostics (pulmonary embolism). 
 
The program will be divided into two phases. Phase 1, a discovery phase to identify putative breath 
biomarker panels and phase 2, a validation phase to replicate and validate selected biomarkers within 
phase 1 using point of care breath sensing devices. 
 
Patient level clinic-pathological and outcome data (spanning the entire acute pathway) will be collected in 
parallel to breath sampling. In addition, breath samples will be acquired during the recovery from the acute 
state and in the stable state post exacerbation to better understand the kinetics of breath analytes 
associated with these diseases.  
 
A parallel cohort of age/home environment matched Adult/ parent/sibling/spousal healthy volunteers will 
also be recruited and evaluated at selected matching time points with breath analysis and a range of 
questionnaires to evaluate factors that are known to influence the metabolome including diet, lifestyle and 
the environment. Additonally patients with stable asthma, COPD and heart failure will be recruited from 
primary and secondary care as disease control populations. 
 
5. OBJECTIVES 
 
5.1 
Primary Objective 
 
To evaluate the sensitivity, specificity, positive and negative predictive value of metabolomic biomarkers in 
exhaled breath samples to identify acute breathlessness,  defined as one or more of (i) patient defiend acute 
breathlessness and/or a (ii) 1 unit increase in Extended Medical Research Council breathlessness score 
(eMRC) 

 
 
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- 
Recruitment in to the acute breathlessness group will be according to the following defined primary 
diagnoses in the discovery phase as defined by the treating physician: 
 
 
(i) Acute Heart failure (n=55-100), (ii) Community acquired pneumonia (n=55-100), (iii) Adult    
exacerbations of asthma and COPD (n=110-200) and (iv) paediatric exacerbations of acute 
childhood wheeze and/or confirmed asthma (n=50-100). Additionally age/home environment 
matched adult/spouse/sibling/parent control subjects (n=55-150 adults/50 children). 
 
 
5.2 
Secondary Objectives 
 
 
To replicate selected metabolomic breath biomarker panels of acute breathlessness using a 
combination of both (i) discovery technologies (including GC-MS) and (ii) point of care auto tuned 
breath sensing devices (including linear IMS, CMS, PTR-MS).  
 
 
To evaluate the primary endpoint stratified by breathlessness as follows (i) change in eMRC by 1 
unit, (ii) patient defined breathlessness and (iii) 100 mm visual analogue score for breathlessness 
 
 
To discover and replicate metabolomic breath biomarkers that differentiate (i) Acute Heart failure, 
(ii) Community acquired pneumonia, (iii) Adult exacerbations of asthma and COPD 
 
 
 
To quantify the level of clinical uncertainty in the primary diagnosis using a 100 mm visual analogue 
scale and independent clinical adjudication of case notes blinded to the following blood biomarkers 
(i) CRP (ii) blood eosinophils (iii) BNP (iv) Troponin-I but not clinical history and chest X-ray.  
 
 
To identify  and discover metabolomic breath biomarkers of acute childhood wheeze and or 
confimed asthma. 
 
To clinically validate metabolomic breath biomarkers generated above using a combination of existing 
pathology markers e.g. BNP, CRP, blood eosinophils and troponin- I and other clinical relevant prognostic 
scoring tools e.g DECAF, CURB65 and Heart failure risk calculator http://www.heartfailurerisk.org/[19]  
- 
Replication of acute breathlessness biomarkers will be according to the following predefined primary 
diagnoses in the replication phase: 
 
               (i) Acute Heart failure (n=55-100),(ii) Community acquired pneumonia (n=55-100), (iii) adult 
 
exacerbations of asthma and COPD (n=110-200),(iv)  paediatric exacerbations of acute childhood 
wheeze and confirmed asthma (n=25-100),(v) age/home environment matched 
adult/spouse/sibling/parent control subjects (n=55-75 adults/25 children) 
 
5.3 Exploratory End Points (where applicable) 
 
1) To evaluate the dynamic profile of breath biomarkers during (i) the acute state, (ii) in the 
recovery state post exacerbation. 

 
 
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2) To evaluate the relationship between breath metabolomic biomarkers and health status/ 
functional measures: 
- 
Health status measures 
- 
Physical performance 
3)  To evaluate the relationship between metabolomic breath biomarkers and clinical outcomes 
including (i) hospital readmission at time points up to 2 years (ii) response to standard clinical 
therapy and (iii) death. 
 
4) To explore and develop breath metabolomic biomarkers of multi-morbidity 
 
5) To evaluate the relationship between diet, lifestyle and environment upon breath metabolomic 
biomarkers.   
 
To generate a biobank of DNA, RNA, urine, plasma, serum and sputum samples for future breathomic and 
other ‘omic molecular pathology studies (including proteomic, transcriptomic, genomic and metabalomic 
studies). 
6 
STUDY DESIGN 
6.1 
Summary of Trial Design 
The clinical study will be a prospective observational study across two acute clinical sites that routinely 
assess and treat cardio-respiratory admissions due to breathlessness within Leicester.  
 
(i) Glenfield Hospital (GGH): Adult cardio-respiratory presentations to Glenfield Hospital (including Clinical 
Decisions Unit (CDU) & inpatient wards), outpatient clinics and NIHR Leicester Biomedical Research Centre 
(BRC) 
(ii) Leicester Royal Infirmary: Adult and paediatric cardio-respiratory presentations to A&E, Children’s 
Assessment Unit (CAU), Diagnostics Development Unit (DDU), inpatient wards, outpatient clinics and 
Research Space   
 
Follow up and stable state controls will be assessed in the outpatient setting within University Hospitals of 
Leicester including the BRC. The study flow is summarised in Figure 1 below. 
  
 
 

 
 
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Figure 1: Study Flow chart 
 
 
Prospective participants with self reported acute breathlessness, either requiring admission or a change in 
baseline treatment, presenting within UHL. The streaming and care pathways at UHL allocate ambulance 
admissions to the two units described above with appropriate clinical care being provided at each site 
according to national guidelines and local protocols / policies.  
Patients with confirmed acute airwary exacerbation or heart failure decompensation as part of other 
research studies at the NIHR Leicester BRC may also be included.  
 
Age- and/or home environment matched adult/spousal/parent and sibling healthy volunteers will be 
recruited where possible on the admissions unit and when not possible in the research units across both 
sites at a separate visit. All attempts will be made to sample breath on at least two of the  same time points 
as patients within these disease groups, however sampling may occur in a different location and at different 
serial time points. All healthy volunteers will complete a range of questionnaires adapted from generic 
cohort studies to characterise their diet, lifestyle and environmental exposures. A stable state control 
population will also be recruited to compare with the unstable state. This will consist of participants with a 
confirmed diagnosis matched with the acute care population but not in the exacerbation state. 
 
Specifically after triage and senior clinical assessment at each of the acute EMBER sites (CDU, DDU, CAU) if a 
primary clinical diagnosis of (i) exacerbation of heart failure, ii) exacerbation of asthma/COPD, (iii)  adult 
community acquired pneumonia or (iv) paediatric exacerbations of acute childhood wheeze and confirmed 
asthma has been made, members of the EMBER research team will evaluate patients using breath diagnosis 
and sensing technologies and additional tools to characterise patients that are acutely unwell. 
 
The following steps  will be employed at all sites:  
1) Identify patients with acute breathlessness defined as one or more of (i) patient defined 
acute breathlessness and/or a (ii) 1 unit increase in (eMRC) 
2) Completeion of study diagnosis at the point of senior clinical decision maker review 
3) Informed consent 

 
 
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4) In selected patients that have not been captured during their acute admission and have 
been discharged prior to EMBER assessments (for example patients that may have been discharged 
rapidly due to the use of an ambulatory care pathway), recall within 48 hours of discharge will be 
perfomed for EMBER assessments 
5) Breath sampling using a standardised CE marked multi-platform breath sampling face mask 
as well as real time breath sampling.  
6) Collection of additional biomarkers for future biomarker discovery campaigns Including a (i) 
urine sample, (ii) blood sample (up to 85 mls) (DNA, RNA ,plasma and serum), peripheral blood cell 
flow cytometry and (iii) spontaneous sputum samples (plugs and supernatants). These samples will 
be collected at time point 1a/1b and at 2 
1) Stable state follow up of all patients will occur following recovery from the acute event. Assessment 
will occur at time of clinical follow up, or as a separate research visit. 
The expected duration of participants within the study will therefore be equivalent to their length of 
hospital stay and up to the stable state follow up visit, with a similar duration for healthy volunteers and 
stable state patients.  
Readmission to hospital within the protocol defined recovery period. Patients that re admit to hospital 
between visits 1 and 2, can have additional 1a 1b assessments. Visit 2 will be taken as recovery following 
the subsequent admission. If a patient is admitted to hospital after visit 2 then they will be eligible to be 
recruited as a new study participant. 
 
Definition of Acute Breatlessness 
A standard definition for acute breathlessness does not exist. We will use the presence of patient reported 
acute breathlessness, above their usual baseline breathlessness  (i.e. in stable state) as a binary variable. 
We will also use the extended MRC score, and 100mm visual analogue score (VAS), as a continuous 
variable.  
The extended MRC score has been validated in chronic disease populations, including in the acute state, 
and may be applied to diseases other than COPD where originaly validated. We will define a change in 
breathlessness of the eMRC as at least a unit change from stable state. In addition a VAS score will be used 
to measure breathlessness.  
 
Definition of Post Exacerbation Recovery  
 
Patient recovery will be defined as  
(i) 
Patient reported recovery from the acute exacerbation spell and back to their baseline 
extended MRC score or clinician defined recovery from the acute exacerbation spell  
and  
(ii) 
At least 6 weeks post exacerbation event (up to 6 months).  
 
 
6.2 
Summary of Additional Assessment and breath sampling procedures/ devices 
A number of analytical devices will be utilised to sample breath in acutely ill patients and based upon the (i) 
clinical site, (ii) operational needs and likely patient grouping (severity of underlying condition at the point 
of admission) within each clinical site, (iii) balance of discovery and point of care sensing technologies: 
breath sensors will be co-located to the two EMBER acute admissions units. All devices will be maintained 
to a high level of operational readiness by the technicians and device specialists within the clinical 
operations group. All devices will be checked for safety by Medical Physics at University Hospitals of 
Leicester NHS Trust. 
 
The following devices will be utilised to evaluate breath volatile organic compounds 

 
 
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 
A portable mass spectroscopy device (Advion Ltd) for discovery based metabolomic breath 
sampling. This device is also capable of real time sampling. 
 
A proton transfer mass spectroscopy device (PTRMS) for discovery based metabolomic breath 
sampling. 
 
A point of care GC-FAIMS Lonestar device (Owlstone Nanotech Ltd) 
 
A point of care linear IMS Bioscout device (Owlstone Ltd: GC-FAIMS Lonestar analyser) 
 
A CE marked breath sampling device developed by Owlstone Nanotech Ltd (Appendix A) will be used to 
sample breath in the majority of patients onto adsorbent Tenax tubes as well as acquiring online 
sampling. This effectively  allows de coupling of the  breath sampling from the breath sensor and 
analysis platforms in selected patients and allows patients that are not able to be mobilised in a chair to 
a real time breath sampling device to be sampled at their bedside. The Owlstone RECIVA sampler 
(Figure 3) is capable of entraining oxygen and is therefore suitable for patients in respiratory failure 
requiring low to moderate flow rates of oxygen to maintain target oxygen saturations. CE marking 
approvals are appended to this protocol in (Appendix B). 
 
Figure 2: Owlstone CE Marked Breath Sampler 
 
 

 
 
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7 
TRIAL PARTICIPANTS 
7.1 
Overall Description of Trial Participants 
 
Each site will recruit patients with acute breathlessness. Final confirmation of the primary indicator 
diagnosis is usually available at discharge (following visit 1b) and after further diagnostics at the stable state 
follow up clinic (at visit 2). The final primary indicator diagnosis and additional comorbidities will be 
recorded at initial senior clinical review, discharge and at follow up. The indicator diagnosis will be used to 
define the number of patients recruited within each disease stratum. 
 
The study will have two distinct phases: 
Phase 1: Discovery of novel breath metabolomic biomarkers. 
Phase 2: Replication and validation of selected biomarkers in Phase 1. 
 
Planned recruitment to meet the primary, secondary and exploratory study endpoints is outlined below. 
 
(i) Acute Heart failure (n=55-100 Phase 1, n=55-100 phase 2)  
(ii) Adult community acquired pneumonia (n=55-100 Phase 1, n=55-10 Phase 2). This condition will be  
defined as new radiological consolidation responsive to treatment according to the British Thoracic Society 
(BTS) community acquired pneumonia guidelines. 
(iii) Adult exacerbations of airway diseases asthma and COPD (n=110-200 Phase 1, n=110-200 Phase 2). This 
condition will be defined as a clinician diagnosis with subsequent or historical evidence of objective 
diagnostic criteria according to national and international guidelines. 
(iv) Paediatric exacerbations of acute childhood wheeze and/or confirmed asthma  (n=55-100 Phase 1, n=25-
50 Phase 2).  
This condition will be defined as a clinician diagnosis and for childhood wheeze/asthma subsequent or 
historical evidence of objective diagnostic criteria according to national and international guidelines. 
(v) Age/environment matched Adult/spousal/parental/ sibling volunteers (n=55-150 adults/50 children) 
discovery phase, (n=55-75 adult/25 children) 
replication phase) with no evidence or history of the indicator disease being evaluated in their 
spouse/sibling/child. 
(vi) Stable state   participants with a confirmed  diagnosis  of  asthma, COPD or heart failure that have not 
had and exacerbation of their disease within the preceding 6 weeks (n=200 (100 in each phase)).   
 
7.2 
Inclusion Criteria  
(i) 
Able to give informed consent for participation in the study. 
(ii) 
Male or Female, aged 16 years or above (adult cohort) and 5-15 years for paediatric patients 
attending the acute care paediatric pathway. 
(iii) 
Capable (in the opinion of the EMBER clinical research investigator(s) of providing serial breath 
samples. 
(iv) 
Diagnosed with acute breathlessness as one of the primary indicator reasons by the clinical 
acute care team. This is not a requirement for healthy subjects or matched controls. 
(v) 
One of the indicator provisional diagnoses identified in section 7.1 following senior review by 
the clinical acute care team. This is not a requirement for healthy subjects or matched controls 
(vi) 
Able (in the Investigators opinion) and willing to comply with all study requirements. 
(vii) 
Willing to allow his or her General Practitioner and consultant, if appropriate, to be notified of 
participation in the study. 
(viii) 
Ability to understand English. 
 
7.3 
Exclusion Criteria 
The participant may not enter the study if ANY of the following apply: 

 
 
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(i) 
Female participants who are known to be pregnant, lactating or planning pregnancy during the 
course of the study. 
(ii) 
Current participation in a clinical trial of an investigative medicinal product or within 3 months 
or 5.5 half-lives of the IMP whichever is longer. 
(iii) 
Active or clinically suspected pulmonary tuberculosis  
(iv) 
In the opinion of the treating physician, breath sampling during the acute admission would be 
clinically unsafe or inappropriate due to the patient’s condition or poor prognosis. Examples 
include malignancy or autoimmune disease with anticipated survival of under 1 year, and 
chronic renal replacement therapy. 
(v) 
Unable or unwilling to give informed consent by visit 1b. 
(vi) 
Any other 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. 
 
8 
STUDY PROCEDURES 
 
A personal legal representative (PeLR) is defined as : A person not connected with the conduct of the trial 
who is suitable to act as a legal representative by virtue of their relationship with the child and available and 
willing to do so.  
 
8.3 Informed Consent 
 
Informed consent in acutely unwell patients (both adults and children) poses a number of potential barriers 
to the conduct of research. However the clinical study team have extensive experience (See Coats TJ et al 
[23]) in consenting both adults and children during acute care settings. During the current study we will 
recruit participants during the acute phase, with full informed consent from the participant or personal legal 
representative (PeLR), in the case of paediatrics. 
 
The patient/PeLR must personally sign and date the latest approved version of the informed consent form 
before any study specific procedures are performed. In the case of paediatric participants they will be asked 
to complete an assent form, in addition to the PeLR consent form. 
 
Written and verbal versions of the participant information and Informed consent will be presented to the 
patient/PeLR  detailing no less than: the exact nature of the study; the implications and constraints of the 
protocol; the known side effects and any risks involved in taking part.  It will be clearly stated that the 
patient is free to withdraw from the study at any time for any reason without prejudice to future care, and 
with no obligation to give the reason for withdrawal. In the event that the participant cannot read the 
information sheet it will be presented verbally only. 
 
All Informed Consent will be obtained by means of participant dated signature and dated signature of the 
person who presented and obtained the informed consent. The person who obtains the consent will 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/parent/legal guardians and a copy retained in the participant 
medical notes.   

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
 
 
8.2 
Screening and Eligibility Assessment 
 
Patients (trial participant groups (i), (ii), (iii), (iv) – see synopsis) will be identified by dedicated member of 
the research team working across both hospital sites following confirmation of the suspected acute 
diagnosis once the patient has been clinically reviewed by a senior clinical decision maker. 
 
The research team will be fully ICH-GCP competent and trained in obtaining informed consent and assent 
according to the hierarchy of consent process outlined in section 8 of this protocol. The research team will 
use available clinical information systems to confirm the nature of previous admissions and indicator 
diagnoses such as the UHL-ICE system, iLAB and the radiology systems for pathology and image viewing 
respectively. Clinical IT systems used to capture clinical meta data are outlined in (Figure 5) below. 
 
Potential participants to the study will be identified shortly after presentation to each of the sites at 
Glenfield Hospital, Diagnostics Development Unit (DDU)/Children's Assessment Units (CAU) at the Leicester 
Royal Infirmary, by members of the healthcare team using the inclusi on criteria of the study. Once the 
decision has been made to admit a patient onto the acute medical unit, potential participants will be 
approached by a member of the healthcare team and asked if they would like to consider taking part in the 
study. If the patient is willing to consider participating in the trial, they will be approached by a member of 
the research team for further information and consent. In many occasions, the healthcare team will also be 
members of the research team. Due to the nature of acute research the research team will identify 
themselves with both the clinical nurse responsible for the patient in question on the admissions unit and 
the clinician(s) responsible so that the consent and screening process can take place in a seamlessness 
manner  and with minimal disruption alongside the patients usual care pathway. 
 
Environment matched adult/spouses/siblings/parents volunteers (trial participants group (v) – see synopsis) 
will be identified and recruited from a number of sources: 
- 
For acute admission the study team will approach the spouse, parent or sibling of the index case and 
seek informed consent for study assessments. All healthy subjects will undergo at last two 
assessments seperated by a duration of up to 6 months to match the acute (visit 1a) and stable state 
(visit 2) time period elapsed in their index case partner/spouse/sibling/child. 
- 
Additional healthy volunteers will be identified from local recruitment databases and via advertising 
 
Additional patients with chronic asthma, COPD and heart failure (trial participants group (vi) – see synopsis) 
will be identified via cardiology and respiratory outpatient clinics at Glenfield Hospital, paediatric respiratory 
outpatient clinics at the Leicester Royal Infirmary, from NIHR Leicester Biomedical Research Centre (BRC) 
patient research databases and will undergo up to 2 visits over a 6 months period. In all cases permission for 
recruitment from outpatients will be sought from the responsible consultant for the patient. The rationale 
for inclusion of this population is to have a stabe state disease control population that have been 
characterised over the same overall reporting period (stable state for measurements and 2 years for 
outcome data collection) as the acute population. 
 
 
 
 
 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
Figure 5: Clinical informatics organogram 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.3 
Study Assessments 
 A summary of both baseline and follow up assessments is outlined in Table 1 (adults) and Table 2 (children) 
below. If appropriate consent is in place the subjects may undertake any combination of any of the 
investigations listed in the investigation table at any of these visits.

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
Table 1: Summary of adult assessments 
Assessments 
COPD 
Asthma 
Pneumonia 
Heart Failure 
Healthy 
Time point 
1a 
1b 
2 
1a 
1b 
2 
1a 
1b 
2 
1a 
1b 
2 
1a 
2 
Written Informed Consent 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
Collection of demographic data (age, gender, height, weight, 
ethnicity, smoking status)* 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
X 
Time of date of admission/discharge# 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
 
 
Breathless Scores* 
 
Patient defined breathlessness 
X^ 
 
X 
X^ 
 
X 
X^ 
 
X 
X^ 
 
X 
X 
X 
Extended MRC dyspnoea score 
X^ 
 
X^ 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
New York Heart Association Dyspnoea Score (NYHA) 
 
 
 
 
 
 
 
 
 
X^ 
 
X^ 
 
 
100 mm visual analogue breathlessness score 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Exacerbation history (previous health care utilisation and 
acute use of medication)* 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
X 
Quality of breath VOCs questionnaire 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Structured Clinical History of presenting complaint and past 
medical history* 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
X 
Assessment of acute disease severity#* 
 
DECAF Score 
X^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
CURB 65 Score 
 
 
 
 
 
 
X^ 
 
 
 
 
 
 
 
BTS Asthma Severity Score 
 
 
 
X^ 
 
 
 
 
 
 
 
 
 
 
Heart failure risk calculator 
 
 
 
 
 
 
 
 
 
X^ 
 
 
 
 
Concurrent medication usage 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X^ 
X 
X 
12 lead ECG* 
X^ 
 
 
X^ 
 
 
X^ 
 
 
X^ 
 
 
 
 
Chest X-ray* 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
 
 
Health Status Questionnaires* 
 
Asthma Quality of Life questionnaire (AQLQ) 
 
 
 
X 
 
X^ 
 
 
 
 
 
 
 
 
COPD assessment tool (CAT) 
X 
 
X^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Protocol: Exhaled Breath Metabolomic Biomarkers in the Acutely Breathless Patient. Final version 4, 1-April 18 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
COPD 
Asthma 
Pneumonia 
Heart Failure 
Healthy 
 
1a 
1b 
2 
1a 
1b 
2 
1a 
1b 
2 
1a 
1b 
2 
1a 
2 
NASA Task Load Index 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Blood tests* 
 
BNP 
X^ 
 
X 
X 
 
 
X 
 
 
X^ 
 
X^ 
X 
 
Troponin-I 
X 
 
 
X 
 
 
X 
 
 
X^ 
 
 
X 
 
CRP 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
X 
Full blood count + differential count 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
X 
DNA and RNA (PAXGENE)~ 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Plasma and serum 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Arterial or capillary blood gas*# 
X^ 
 
 
X^ 
 
 
X^ 
 
 
X^ 
 
 
 
 
Peripheral blood flow cytometry  
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Lung function test 
 
Spirometry* 
X^ 
 
X^ 
X^ 
 
X^ 
 
 
 
 
 
 
X 
X 
Oscillometry* 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Exhaled nitric oxide (FeNO) 
 
 
 
X 
 
X^ 
 
 
 
 
 
 
 
 
Sputum sample (spontaneous^ or induced) 
X^ 
 
X 
X^ 
 
X 
X^ 
 
X 
X^ 
 
X 
X 
X 
Volatile organic compounds (VOCs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine sample* 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
Echocardiography* 
X 
 
 
 
 
 
 
 
 
X^ 
 
 
 
 
Quadriceps ultrasound 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
4 meter gait speed 
 
X 
X 
 
 
 
 
 
 
 
X 
X 
 
 
Review of Adverse Events & Serious Adverse Events 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
Physical Exam* 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X^ 
 
X^ 
X 
 
*Can be performed at time points 1a or 1b. for healthy volunteers this can be completed at visit 1 or 2. For informed consent see protocol, ^ may be part of routine 
clinical care according to UHL Trust Guidelines. Arterial, capillary blood gases and blood cultures only done in selected clinical cases, # Acute patients only ~If DNA 
taken at time point 1a will not be repeated at timepoint 2. 
 
 
 

 
 
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Table 2: Summary of children assessments 
Assessments 
Childhood Wheeze 
/Confirmed Asthma 
Healthy 
Time point 
1a 
1b 
2 
1a 
2 
Written Informed Consent 
X 
 
 
X 
 
Collection of demographic data (age, gender, height, weight, ethnicity, smoking status)* 
X^ 
 
X^ 
X 
X 
Time of date of admission/discharge# 
X^ 
X^ 
 
 
 
100 mm visual analogue breathlessness score* 
X 
 
X 
X 
X 
Exacerbation history (previous health care utilisation and acute use of medication)* 
X^ 
 
X^ 
X 
X 
Quality of breath VOCs questionnaire 
X 
X 
X 
X 
X 
Structured Clinical History of presenting complaint and past medical history* 
X^ 
 
X^ 
X 
X 
Concurrent medication usage 
X^ 
X^ 
X^ 
X 
X 
Chest X-ray* 
X^ 
 
X^ 
 
 
Health Status Questionnaires* 
 
Asthma control questionnaire (ACQ) 
X^ 
 
X^ 
 
 
Asthma quality of life questionnaire (AQLQ) 
X^ 
 
X^ 
 
 
NASA Task Load Index 
X 
X 
X 
X 
X 
Blood tests*+$ 
 
CRP 
X^ 
 
X^ 
X 
X 
Full blood count + differential count 
X^ 
 
X^ 
X 
X 
DNA and RNA (PAXGENE) 
X 
 
X 
X 
X 
Plasma and serum 
X 
 
X 
X 
X 
Arterial or capillary blood gas* 
X^ 
 
 
 
 
Blood culture* 
X^ 
 
 
 
 
Lung function test 
 
Spirometry* 
X^ 
 
X^ 
X 
X 
Exhaled nitric oxide (FeNO) 
X 
 
X 
X 
X 
Sputum sample (spontaneous^ or induced)$ 
X^ 
 
X^ 
X 
X 
Volatile organic compounds (VOCs) 
X 
X 
X 
X 
X 
Urine sample* 
X 
 
X 
X 
X 
Review of Adverse Events & Serious Adverse Events 
 
X 
X 
 
X 
Physical Exam* 
X^ 
 
X^ 
X 
X 
*Can be performed at time points 1a or 1b. For informed consent see protocol, ^ Part of routine clinical care as part of  the University Hospitals of Leicester, acute 
care  clinical pathway. Will be performed in selected clinical cases based on clinical need, # Acute patients only, + up to 35 mls maximum per visit, $ optional

 
 
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A description of the research assessments is outlined below 
 
 
Demographics 
All standard clinical demographics routinely recorded in acutely unwell patients will be captured using study 
specific case report forms (CRFs). These will include the age, sex, height, weight, smoking status and 
ethnicity of the patient.  
 
 
Medical History 
All standard clinical data routinely recorded in acutely unwell patients will be captured using study specific 
case report forms (CRFs). This will include co morbidities usually supplied with the GP referral letter and the 
use of hospital clinical information systems such as ICE to cross check co morbidities where necessary using 
discharge summaries as well as case notes review. 
 
 
Concomitant Medication  
Current medication will be collected. This will comprise of usual medication, medication in the two weeks 
prior to visit 1a .In the case of admission, additional confirmation  will be performed after standard 
pharmacy review by the admissions pharmacist allocated to the admissions unit in question. 
 
 
Physical Examination 
All standard clinical data routinely recorded in acutely unwell patients will be captured using study specific 
electronic case report forms (CRFs). Standard clinical examination findings relevant to the indicator diagnosis 
in question will be recording and will include pulse, blood pressure, temperature, respiratory rate, oxygen 
saturations and impairment of cognition. 
 
 
Collection and recording of standard clinical care pathway diagnostic 
The majority of acute care admissions with undifferentiated breathlessness will undergo various 
combinations of the following assessments dependent upon their admission diagnosis. 
- Chest x ray 
- 12 lead ECG  
- Laboratory blood tests may include: Urea and electrolytes, liver function tests, albumin, corrected 
calcium,phosphate, D-Dimer, BNP, Troponin-I, full blood count, haematocrit and differential white cell count. 
- Additional investigation such echocardiography (will be done during acute admission between time points 
1a and 1b). Echo will measure LV and RV function, LA size, vavular function, LV wall thickness and e/e' on 
tissue Doppler. 
 
**The responsibility to act upon and record abnormal investigations within the patient’s clinical notes will 
remain with the admitting clinical team who will have ordered and requested the investigations. 
 
 
Breath sampling procedures (PLEASE REFER TO  APPENDIX D: ASSET ALLOCATION APPENDIX FOR A 
DETAILED SUMMARY OF THE CORE BREATH DETECTION TECHNOLOGIES) 
Breath sampling will be performed using various combinations of the devices below dependent upon 
patient location, level of illness (acuity) and phase of the program (discovery/replication). Breath sampling 
will be limited to a duration acceptable to a patient (typically 30 mins) at any particular study visit to 
minimise disruption to the patient care pathway and flow. 
 
 
 

 
 
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Advion CMS: Discovery Phase (Phase 1) Breath Analysis: 
The Advion Expression compact mass spectrometer (CMS) is a single quadrupole mass spectrometer that 
enables the study of volatiles in breath by adding a simple modification at the front end of the instrument. 
The volatiles are drawn into the mass spectrometer ion source directly from the patient’s breath using a 
Venturi pump. This pump creates a Venturi effect sucking the volatiles into the mass spectrometer at a rate 
of approximately 300 mL/min. When sampling, patients wear a facemask which is fed with approximately 
35 l/min of purified air to which the Venturi pump inlet is connected. The extracted breath samples pass 
through a transfer line heated to an optimal temperature to avoid condensation of the volatiles. The 
minimum time required to perform a breath sample is 2 min. After this sampling time the data can be 
extracted immediately for analysis. 
 
GC-IMS (B&S Analytiks): Replication Phase (Phases 2) Breath Analysis: 
Gas chromatography ion mobility spectrometry (GC-IMS) enables the detection of VOCs in exhaled breath 
of patients in trace gas concentrations (ng/L-pg/L range). Sampling takes place using a Spiroscout 
spirometer. The patients exhale through a disposable mouth piece connected to a Teflon tube. A 
piezoelectric pressure sensor is used to monitor the breathing profile, this opens the sampling valve at the 
appropriate point in the breath profile to collect end-tidal breath in a sample loop of 10 mL volume. After 
filling this loop, the collected sample air is then transferred to a multicapillary column for a 
chromatographic separation, which is achieved in 12 min. The separated molecules are then transferred 
into the IMS, ionised and then separated  according to their mobility in a weak electric field. 
 
GC-FAIMS (Owlstone Nanotech Ltd): Replication Phase (Phase 2) Breath analysis: 
Gas chromatography field asymmetric ion mobility spectrometry (GC-FAIMS) is a gas detection technology 
that separates and identifies chemical ions based on their mobility under a varying electric fields at 
atmospheric pressure. Samples are collected on adsorbent tubes (Tenax/Carbograph) using a CE marked 
handheld sampling device. Samples are then extracted from tubes using thermal desorption and then 
analysed by GC-FAIMS. The combination of the high selectivity of GC with the high sensitivity of FAIMS 
enables the detection of volatiles in a wide range of matrices such as aqueous, solid and gaseous. FAIMS is 
also known as differential mobility spectrometry (DMS). 
 
Real time breath monitoring using proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS): 
In order to sample a patient’s breath using this technique, two breath sampling devices will be used. The 
first device is a Loccioni SOFIA GSI-S; the subject is required to exhale a single breath, five times (three if 
providing five samples proves too difficult) into a sterile mouthpiece connected to an electrostatic 
bacterial/viral filter whilst wearing a nose clip (all CE marked). Flow from the mouthpiece passes into a gas 
sampling interface capnograph (Loccioni GSI-S – CE marked) and real-time user feedback of flow is provided 
on screen, allowing the regulation of the breath sampling rate. The gas sampling interface acts to 
simultaneously trigger the acquisition of the PTR-ToF-MS data and the exhaled breath travels through the 
capnograph down a heated sample line into the ion source of the PTR-ToF-MS 
The second breath sampling device is a ReCIVA breath sampler (Owlstone), as described in Section 6.2, with 
one of the adsorbent Tenax tubes replaced with an outlet tube adapted for online sampling. The exhaled 
breath is transferred to the PTR-ToF-MS via a heated transfer line connected to the outlet tube, 
continuously drawn at a constant flow rate by the PTR-ToF-MS. The online adaptation of the consumable 
adsorbent tube does not affect the CE mark of the ReCIVA sampling device. 

 
 
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Once the breath sample reaches the PTR-ToF-MS, via either breath sampler, the breath mixes with 
protonated water (H3O+) inducing proton transfer to the target volatile organic compounds (VOCs) present, 
resulting in their ionisation.  Sample ions are then guided into the time of flight mass spectrometer and 
mass spectra, showing the abundance and mass of the VOCs present, are collected throughout the 
exhalation. Following sampling, mouthpieces, filters and nose clips are disposed of and all patient 
contacted surfaces wiped down with antiseptic cleaning wipes in preparation for the next patient. 
 
 
Sputum collection 
Spontaneous or induced sputum samples can be collected.  In the case of induced sputum 
sample collection all subjects will have a baseline FEV1 measured.  Sputum collection will be 
collected using the principles as defined by the European Respiratory Society Guidelines.  All 
equipment used for performing the following procedure will be de-contaminated according to 
local working instructions. The procedure may be stopped prematurely if: (1) the subject’s FEV1 
drops by greater than 20% of the post-salbutamol FEV1 baseline value, (2) the subject feels any 
discomfort and does not want to continue with saline inhalation procedure or (3) the investigator 
feels that it is unsafe to continue with the saline inhalation. Sputum supernatants will be stored at 
the investigative site in dedicated -80 C freezers. 
 
 
Urine Collection 
Urine will be collected from subjects. It will be collected into a suitable container and stored at 
the investigative site in dedicated -80 C freezers. 
 
 
Lung function 
Lung function testing will include spirometry (pre and post bronchodilator), FeNO (Fraction exhaled Nitric 
Oxide), hand held oscillometry (adults asthma patients only). Peak flow measurements (to be recorded as 
part of routine care in asthmatics), and volatile breath analysis. All of these are non-invasive clinical tests. 
 
 
Research Echocardiography (ECHO) 
For patients (excluding healthy volunteers) where there is no clinical indication for ECHO a research ECHO 
will be performed by a certified technician following written informed consent from the patient any time 
point between visit 1a and visit 1b. 
 
 
Blood sampling 
In addition to the routine clinical blood samples will be taken for RNA and DNA, serum and plasma, 
peripheral blood cell flow cytometry analysis per visit. This may total up to 85 mls for an adult and 35mls for 
a child.  Samples will be barcoded and stored at the investigative site in dedicated -80 C freezers. Samples 
may be transferred to the NIHR national bio sample centre subject to available funding.  Blood samples will 
be taken at the same time point as the clinical samples to minimise inconvenience to the patient. 
 
 
Health Questionnaires 
Questionnaires regarding the subjects’ health and disease status will be completed. These will 
include measures of health status.  
 
 
NASA Task Load Index 
It is a widely used, subjective, multidimensional assessment tool that rates perceived workload in order to 
assess a task, system, or team's effectiveness or other aspects of performance. It assesses work load on five 
7-point scales including mental demand, physical demand, temporal demand, performance, effort and 

 
 
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frustration. Increments of high, medium and low estimates for each point result in 21 gradations on the 
scales. This tool will be incorporated into EMBER to assess the overall performance and feasibility of using 
ReCIVA mask in breath collection.  
 
 
Physical performance 
4 meter gait speed (the time it takes to walk 4 meters) and quadriceps size measured using 
ultrasound will be performed to assess physical performance in selected patients. Both are measures of 
frailty, non-invasive, and validated in the acute care setting in chronic respiratory disease. If ultrasound 
measure is missed at timepoint 1 then it will not be taken at timepoint 2. 
 
8.6  
Definition of End of Study 
 
The end of trial is the date of the last patient last visit (Visit 2). 
 
Each participant will have 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) 
● Pregnancy 
● Significant protocol deviation 
● A serious adverse event which requires results in inability to continue to comply with 
study procedures 
● Disease progression which results in inability to continue to comply with study procedures 
● Consent withdrawn 
● Lost to follow up 
 
 
The reason for withdrawal will be recorded in the CRF.   
 
8.7 
Source Data 
In this study a paper CRF will be collected for all ‘ research only‘ measurements defined in tables 1 and 2. 
The electronic CRFs will be used as the source document for the captured data fields described in Table 1 
and 2 that are part of routine clinical care. The rationale for this is that data collection for routine clinical 
care measures within MRC EMBER is populated in to the eCRF from existing UHL IM&T systems (Figure 4 e.g. 
IMPAX, ilab, CODE BREAKER) as well as prospectively confirmed by the research team for relevant study 
clinical meta data of interest e.g. smoking status, medication. 
 
9. SAFETY REPORTING 
9.1 
Definitions 
 
9.1.1 
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 the study, whether or not considered related to the 
study. 

 
 
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9.1.2 
Adverse Reaction (AR) 
All untoward and unintended responses related to the study. 
All cases judged by either the reporting medically qualified professional or the sponsor as having a 
reasonable suspected causal relationship to the study qualify as adverse reactions.   
 
9.1.3 
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. 
 
9.1.4 
Serious Adverse Event or Serious Adverse Reaction 
A serious adverse event or reaction is any untoward medical occurrence that: 
 
Results in death, 
 
Is life-threatening, 
NOTE: The term "life-threatening" in the definition of "serious" refers to an event in which the participant 
was at risk of death at the time of the event; it does not refer to an event which hypothetically might have 
caused death if it were more severe. 
 
Requires inpatient hospitalisation or prolongation of existing hospitalisation, 
 
Results in persistent or significant disability/incapacity, or 
 
Is a congenital anomaly/birth defect. 
 
Other important medical events* 
*Other events that may not result in death, are not life threatening, or do not require hospitalisation, may be 
considered a serious adverse event when, based upon appropriate medical judgement, the event may 
jeopardise the patient and may require medical or surgical intervention to prevent one of the outcomes listed 
above. 
 
9.1.5 
Expected Serious Adverse Events/Reactions 
The patient cohort being studied are frail and have a high hospital readmission rate and mortality rate. For  
example in those with a diagnosis of COPD both national and local data suggest readmission to hospital will 
be 20% at one month, 33% at 3 months and 60% at one year. A number of these patients will be 
readmitted on more than one occasion (our study Greening N et al, BMJ 2014) demonstrated 599 
hospitalisations in one year in a similar COPD population of 389. In addition mortality is expected to be 14% 
at 3 months and 20% at one year.  
 
Given this is an observational study and the rates that are expected we would reasonably expect that 
hospitalisation or death are not routinely reported as SAEs (we would anticipated an excess of 600 SAEs if 
not). Therefore expected serious adverse events include (i) clinical deterioration and death of acutely 
breathless patients, (ii) adverse drug reactions to pharmacotherapy administered during the patients 
clinical care pathway, (iii) readmission to hospital for acute breathlessness due to acute exacerbations of co 
morbid conditions during the study period. 
 
However, if any adverse events are experienced during any of the measures performed specifically related 
to research procedures (Table 1 and 2) e.g. breath sampling, administration of questionnaires, then these 
will be reported using the approved reporting procedure. 

 
 
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9.1.6 
Suspected Unexpected Serious Adverse Reactions 
A serious adverse reaction, the nature or severity of which is not consistent with the applicable product 
information  
 
9.2 
Reporting Procedures for All Adverse Events 
All AEs occurring during the study observed by the investigator or reported by the participant, whether or 
not attributed to study, will be recorded on the CRF.   
The following information will be recorded: description, date of onset and end date, severity, assessment of 
relatedness to study, other suspect device and action taken.  Follow-up information should be provided as 
necessary.  
AEs considered related to the study as judged by a medically qualified investigator or the sponsor will be 
followed until resolution or the event is considered stable.  All related AEs that result in a participant’s 
withdrawal from the study or are present at the end of the study, should be followed up until a satisfactory 
resolution occurs. 
It will be left to the investigator’s clinical judgment whether or not an AE is of sufficient severity to require 
the participant’s removal from the study.  A participant may also voluntarily withdraw from treatment due 
to what he or she perceives as an intolerable AE.  If either of these occurs, the participant must undergo an 
end of study assessment and be given appropriate care under medical supervision until symptoms cease or 
the condition becomes stable. 
The severity of events will be assessed on the following scale:  1 = mild, 2 = moderate, 3 = severe.   
The relationship of AEs to the study will be assessed by a medically qualified investigator.  
 
9.3 
Reporting Procedures for Serious Adverse Events 
All SAEs, except those expected ones defined in section 9.1.5 that do not require immediate reporting (see 
9.1.5), must be reported to the Sponsor within 24 hours day of discovery or notification of the event.  The 
Sponsor will perform an initial check of the information and ensure that it is reviewed at the next R&D 
Management meeting.  All SAE information must be recorded on an SAE form and sent to the Sponsor 
using the appropriate reporting form and the contact details on there. Additional information received for a 
case (follow-up or corrections to the original case) needs to be detailed on a new SAE form which must be 
sent to the Sponsor using the appropriate reporting form and the contact details on there.  
The Sponsor will report all SUSARs to the Research Ethics Committee concerned. Fatal or life-threatening 
SUSARs must be reported within 7 days and all other SUSARs within 15 days. The CI will inform all 
investigators concerned of relevant information about SUSARs that could adversely affect the safety of 
participants. 
In addition to the expedited reporting above, the CI shall submit once a year throughout the study or on 
request an Annual Report to the Ethics Committee which lists all SAEs / SUSARs that have occurred during 
the preceding 12 months. 
 
 
10.  STATISTICS 
10.1 Description of Statistical Methods (Detailed statistical anad analytical methods are outlined 
in the  MRC-EMBER data analysis plan). 
 
Primary Analysis 
For the primary analysis the outcome is a nominal variable with levels (1) Acute breathlessness (2) Recovery 
form acute breathlessness. The relationship between the primary outcome and the metabolomics 
biomarkers in exhaled breath sample will be modelled using multinomial logistic regression. Acute 

 
 
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breathlessness will be defined as one or more of (i) patient defined acute breathlessness and/or a (ii) 1 unit 
increase in Extended Medical Research Council breathlessness score (eMRC). In addition to metabolomics 
markers the following independent variables will be included in the model: age, sex, standard acute care 
pathology markers blood eosinophils, BNP, CRP, Troponin-I,  canonical VOCs extracted from breath analysis 
and where relevant validated co morbidity scores (e.g. Charlson comorbidity score). 
 
Adjudication 
 
All acute/healthy adult patients’ notes will be sequentially divided. These will then be reviewed and 
independently adjudicated by two independent respiratory consultants, blinded to admission bloods and 
diagnosis. The panel will independantly decide the primary diagnosis as well as mark their level of certainty 
on a 100 mm VAS scale. The panel member will then be able to review imaging, ECGs, and ABGs but not 
blood results. Results will be both -validated by separate panel member to ensure agreement using Bland-
Altman and Correlations and within rater agreement will be assessed by replicate analysis of at least 20  
cases 4 weeks apart.   
 
 
Binary logistic regressions will be utilised to assess associations between VOCs and acute disease. ROC 
curves will be produced for individual predictors in the primary analysis. Additional techniques for multi 
variate analysis including but not limited to cluster analysis, structural equation modelling and topological 
data analyses will be applied where relevant. Tables of descriptive statistics will be compiled for all key 
variables. All analysis will be performed using SAS 9.4 http://www.sas.com and R 3.2.0 https://www.r-
project.org/ . 
 
Secondary Analysis 
For the secondary analysis pertaining to other definitions of acute breathlessness, visual analogue 
breathlessness scores (100mm) and a 1 unit increase in the extended medical research council analysis 
outlined above for the primary endpoint will be performed based upon stratification of breathlessness type. 
. 
For the secondary analyses pertaining to discovery of breath metabolomics markers the outcome is a 
nominal variable with levels (1) Acute Heart failure (2) Community acquired pneumonia 3) Adult 
exacerbations of asthma/COPD (both in combination and considered individually) and (4) Acute 
exacerbations childhood wheeze and confirmed asthma in children. The relationship between the primary 
outcome and the metabolomics biomarkers in exhaled breath sample will be modelled using multinomial 
logistic regression and ROC curves. In addition to metabolomics markers the following independent variables 
will be included in the models where necessary (improve model fit): age, sex, validated co morbidity scores 
such as the Charlson score,  indicator disease risk scores such as the CURB-65 pneumonia score, DECAF 
COPD exacerbation scores and treatments commonly used acutely for the indicator diagnoses. 
 
Subsidiary Analysis 
For subsidiary analyses modelling risk of re admission, death and length of stay we will utilise the following 
approaches 
- 
Cox proportional hazards model will be fitted and competing risk models will be fitted for 
readmission. Logistic regression analysis will be used when looking at risk at particular time points 
for readmission.  
- 
The number of exacerbation events will be modelled using Poisson or Negative Binomial regression. 
- 
Relationship between death and breath biomarkers will be evaluated using a logistic regression 
model.  

 
 
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For subsidiary analyses pertaining to dynamic profile of breath biomarkers during (i) the acute states (time 
points 1a), (ii) transition to the stable state (time point 1b) and (iii) in the chronic state 8-12 weeks post 
exacerbation (time point 2), a repeated measures model with a random intercept and random effect for 
time will be fitted, the random effects will be fitted for each patient. For the repeated measures mixed 
model an unstructured covariance will be assumed.  
Other outcomes are exploratory and based around feasibility of an acute care platform for airways disease. 
Statistical analyses for these outcomes will be performed as appropriate for hypotheses derived in this 
exploration. 
10.2 The Number of Participants 
The sample size for the study is based on the requirements of the multinomial logistic regression model for 
the primary analysis. We estimate that if the number of events to number of variables in the model is in the 
ratio 20:1 a sufficiently large sample size will be obtained[24]. We regard this study as being exploratory it will 
serve as a basis for determining the sample size required for a larger study in the future. (Appendix D) 
 
10.3 The Level of Statistical Significance 
All tests will be performed at the 5% significance level. 
 
10.4 Criteria for the Termination of the Trial. 
Last participant, last visit 
 
10.5 Procedure for Accounting for Missing, Unused, and Spurious Data. 
 
Describe 
A table listing all key variables with percentage of missing data will be reported and where possible the 
reason for missing data will be reported. Missing data will be imputed using PROC MI in SAS. Spurious data 
will be reported and where possible correct values will be inserted, if it is not possible to supply correct 
values value will be deleted.  
 
10.6 Procedures for Reporting any Deviation(s) from the Original Statistical Plan 
An interim sample size calculation will be performed at the end of the discovery phase (Quarter 2, year 3) of 
the project; this calculation will identify the optimal number of patients in each replication phase disease 
indicator group based upon the optimal number of metabolomic biomarkers associated with the disease and 
the key outcome data from the discovery phase e.g. risk prediction, 30 / 60 day re admissions, discrimination 
of disease state from healthy state.  
 
10.7 Inclusion in Analysis 
All eligible participants, that have provided informed consent and participated in either discovery or 
replication phase studies. 
 
11. 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. 
 
12. 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.  

 
 
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Internal  monitoring by the research team within the BRC will be performed according to ICH GCP. Data will 
be evaluated for compliance with the protocol and accuracy in relation to source documents. Following 
written standard operating procedures, we will verify that the clinical study is conducted and data are 
generated, documented and reported in compliance with the protocol, GCP and the applicable regulatory 
requirements.  
The EMBER clinical operations group and clinical work stream group (WP4) described in section 15 of this 
protocol will meet fortnightly and monthly respectively to discuss overall quality control and assurance of 
the clinical study. In addition the University of Leicester operates a risk based audit programme to which 
this study will be subject to. 
13. CODES OF PRACTICE AND REGULATIONS 
13.1 Ethics 
The study will obtain ethical approval from an approved ethics committee and be registered on a trial 
database. 
 
13.2 Sponsor Standard Operating Procedures 
All relevant Sponsor SOPs will be followed to ensure that this study complies with all relevant legislation 
and guidelines  
 
13.3 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). 
 
13.4 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. 
 
13.5 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) 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.    
 
13.6 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.   
 
13.7  Other Ethical Considerations 
The key ethical consideration specific to the MRC-EMBER study is the recruitment of acutely unwell patients 
that may not be able to provide informed consent. A hierarchical consent process will be adopted for the 
study with gold standard always being written patient informed consent at time point 1a, whilst accepting 
that for some patients and children personal legal representatives may be required to give written infirmed 
consent pending patient consent. In all cases written consent from the patients or parent in the case of 
young children will be  obtained prior to time point 1b assessments.  
 

 
 
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14. DATA HANDLING AND RECORD KEEPING 
EMBER study number 
The participants in the study will be assigned a EMBER study number. This number will identify the 
participant through all stages of data collection and integration.  
To reduce errors the EMBER study number will also contain a check digit and it will also be printed as a 
barcode whenever appropriate, so it can be scanned into data collection tools. 
 
e-CRF data 
 
The EMBER clinical data platform will be built using a bespoke informatics platform (ClinicLE) used previously 
in respiratory research [18]. 
 
Several features of this platform will ensure high quality data collection and integration including: 
- 
user access control so users have permissions to enter/view only appropriate data forms 
- 
integration of data from hospital systems including patient demographics, pathology test results and 
respiratory physiology 
- 
data field validation (allowing specific types of data; specific ranges of values; or a specific set of 
selectable options) 
- 
appropriate field design (e.g. using Y/N options instead of checkboxes to ensure the answer is not 
ambiguous) 
- 
verification through e-signatures and record locking capability 
- 
regular execution of data quality rules to ensure missing/incorrect data is identified quickly 
- 
branching logic so that fields which are not relevant are not displayed to the user 
- 
a form design and data entry audit log/history 
 
A formal test plan will be written during the implementation of the e-CRFs and clarified with other 
collaborators. Following the completion of the e-CRF implementation, the test plan will be executed to 
ensure that data collection forms operate within the expected parameters. 
A Standard Operating Procedure will be written documenting the use of the EMBER system, specifically for 
the purpose of entering and managing e-CRF data by the EMBER research staff. 
The participants’ name and any other identifying detail including date of birth will NOT be included in the 
final exports of e-CRF data used for research purposes.  
 
Clinical data 
 
Identifiable clinical data exports will be de-identified programmatically by: 
- 
populating the EMBER study number from the recruitment database  
- 
removing hospital numbers, NHS numbers and other patient identifiers 
- 
removing name, address and GP details 
- 
removing or shifting date values 
- 
filtering out free text which may contain identifiable information 
 
Experimental data 
Data generated in laboratory experiments will be linked to EMBER study participants using the EMBER study 
number. No other identifiable data will be present in this data. 
 
Data storage and backup 
EMBER clinical data will be stored in an access-controlled database back-end located at the University 
Hospitals of Leicester. The data will only be available through the hospital network, via the EMBER web 

 
 
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application front-end. The database will be backed up informally every hour to a UHL shared drive in a 
process managed by the hospital’s IT department. 
Experimental data and subsequent analyses will be stored using the University of Leicester’s research data 
storage (R) drive backup system (http://www2.le.ac.uk/offices/itservices/ithelp/services/rfs) will be used to 
maintain all raw experimental data and subsequent analyses. 
 
Risks 
Unauthorised access to data: Users will be given appropriate access to databases/data shares for their role, 
based on recommendations by the PI and other investigators. Identification of research subjects: Research 
subjects will be identified only through their EMBER study number. The connection between this ID and the 
patient’s demographics will only be available to authorised personnel. Data corruption: In the unlikely event 
that data becomes corrupted, it will be restored from a backup of the server which is less than 24 hours old. 
In the case of the EMBER database this means a small amount of data may be missing, and it will have to be 
re-entered. To mitigate this, paper copies of CRFs should be kept for at least a week after entry into the 
EMBER clinical data platform. Erroneous data entry: Data entered from paper forms will be verified and if 
required corrected by a separate person. Data records will then be electronically signed and locked. 
 
EXTERNAL DATA SHARING 
 
Data and samples may be analysed by other investigators/organisations collaborating with the study 
investigators. In such cases, patient confidentiality will be strictly maintained. 
 
 
15. STUDY GOVERNANCE 
 
15.1 Clinical Steering Committee (TSC) 
The clinical study will be managed through the clinical work package (WP4) of the MRC-EMBER molecular 
pathology node. The core clinical group (Professor Siddiqui, Dr Pandya, Dr Greening and Professor Coates) 
all have membership within the EMBER Executive steering group. 
 
The Executive Group comprises the work strand leads and other key PIs. A Scientific Strategy Board 
comprising all Co-PIs and industrial partners will guide the Executive Board. An operations group chaired by 
the EMBER's manager will oversee delivery of work. An External Advisory Board with membership derived 
from experts in the field,  Leicester and Loughborough Universities and NHS Research Directors will also 
oversee the clinical delivery and progress. The Sponsor will be sent minutes from the Scientific Strategy 
Board and External Advisory Board Meetings. An overview of the governance procedure within EMBER that 
the clinical work package (WP4) contributes towards is outlined in Figure 6 below. 
 
 
 

 
 
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Figure 6: MRC EMBER program management organogram 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
15.2 Data Safety Monitoring Committee (DSMC) 
 
The operations group within EMBER will report any unexpected serious adverse events to the core clinical 
group (Professor Siddiqui, Dr Gaillard Dr Greening and Professor Coats). All unexpected SAEs will be 
discussed by the core clinical group at monthly EMBER executive board meetings. 
 
16. FINANCING AND INSURANCE 
Direct Research Costs:  Direct research costs required to deliver the stated research: including research 
staff, technicians, breath sampling, breath analysis and bioinformatics are fully funded by the UK Medical 
Research Council (MRC) and The Engineering and Physical Sciences Research Council (EPSRC) grant number 
MR/N005880/1. Additional funding has been received from the Midlands Asthma and Allergy Research 
Association (MAARA) and the Leicester National Institute for Health Respiratory (NIHR) Biomedical Researh 
Unit, for the EMBER clinical program. 
 
NHS Treatment Costs: Not applicable for acute research studies as by definition patient care will often 
continue after the R&D activity has ceased due to the fact that patients will enter chronic care pathways.  
 
NHS Support Costs: None anticipated as the research in question is a discovery biomarker campaign. 
 
Insurance will be provided by the University of Leicester. 
 

 
 
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17.  PUBLICATION POLICY 
Publications will be prepared according to the MRC-EMBER consortium agreement and the  University of 
Leicester publications policy. In brief all intended publications will be submitted to the EMBER executive 
board for review and comments within 60 days of journal submission. Authorship will be according to 
contribution and internationally recognised guidance on journal authorship. 
18. REFERENCES 
1. Green SM, Martinez-Rumayor A, Gregory SA, Baggish AL, O′Donoghue ML, Green JA, et al. Clinical 
uncertainty, diagnostic accuracy, and outcomes in emergency department patients presenting with 
dyspnea. Arch Intern Med 2008;168:741-8. 
2. Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, et al. Rapid measurement of 
plasma B-type natriuretic peptide in the emergency diagnosis of heart failure which possibly reflects the 
improvement in cardiac function secondary to treatment. N Engl J Med 2002;347:161-7 
3. Januzzi JL, van Kimmenade R, Lainchbury J, Bayes-Genis A, Ordonez-Llanos J, Santalo-Bel M, et al. NT-
proBNP testing for diagnosis and short-term prognosis in acute destabilized heart failure Collaborative of 
NT-proBNP Study. Eur Heart J 2006;27:330-7. 
4. Bafadhel M, Clark TW, Reid C, Medina MJ, Batham S, Barer MR, Nicholson KG, Brightling CE. procalcitonin 
and C-reactive protein in hospitalized adult patients with community-acquired pneumonia or exacerbation 
of asthma or COPD. Chest;139(6):1410-8. 
5. Bafadhel M, McKenna S, Terry S, Mistry V, Pancholi M, Venge P, Lomas DA, Barer MR, Johnston SL, 
Pavord ID, Brightling CE. Blood eosinophils to direct corticosteroid treatment of exacerbations of chronic 
obstructive pulmonary disease: a randomized placebo-controlled trial. Am J Respir Crit Care Med. 2012 Jul 
1;186(1):48-55. 
6. van Vugt SF, Broekhuizen BD, Lammens C, Zuithoff NP, de Jong PA, Coenen S, Ieven M, Butler CC, 
Goossens H, Little P, Verheij TJ; GRACE consortium. Use of serum C reactive protein and procalcitonin 
concentrations in addition to symptoms and signs to predict pneumonia in patients presenting to primary 
care with acute cough: diagnostic study. BMJ. 2013 Apr 30;346:f2450 
7. Steer J, Gibson J, Bourke SC. The DECAF Score: predicting hospital mortality in exacerbations of chronic 
obstructive pulmonary disease.Thorax. 2012 Nov;67(11):970-6.  
8. Chen JH, Chang SS, Liu JJ, Chan RC, Wu JY, Wang WC, Lee SH, Lee CC. Comparison of clinical 
characteristics and performance of pneumonia severity score and CURB-65 among younger adults, elderly 
and very old subjects.Thorax. 2010 Nov;65(11):971-7 
9. Probert C.S., Ahmed I., Khalid T., Johnson E., Smith S., Ratcliffe N. Volatile organic compounds as 
diagnostic biomarkers in gastrointestinal and liver diseases. J. Gastrointestin. Liver Dis. 2009;18:337–343.  
10. Fens N., van der Schee M.P., Brinkman P., Sterk P.J. Exhaled breath analysis by electronic nose in 
airways disease. Established issues and key questions. Clin. Exp. Allergy. 2013;43:705–715.  
11. Patel K. Noninvasive tools to assess liver disease. Curr. Opin. Gastroenterol. 2010;26:227–233.  
12. Schubert J., Miekisch W., Geiger K., Nöldge-Schomburg G. Breath analysis in critically ill patients: 
Potential and limitations. Expert Rev. Mol. Diagn. 2004;4:619–629. 
13. Samara MA, Tang WH, Cikach F Jr, Gul Z, Tranchito L, Paschke KM, Viterna J, Wu Y, Laskowski D, Dweik 
RA. Single exhaled breath metabolomic analysis identifies unique breathprint in patients with acute 
decompensated heart failure. J Am Coll Cardiol. 2013 Apr 2;61(13):1463-4.  
14. Fowler SJ, Basanta-Sanchez M, Xu Y, Goodacre R, Dark PM.Surveillance for lower airway pathogens in 
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2015 Apr;70(4):320-5.  
15. Fens N, van der Schee MP, Brinkman P, Sterk PJ. Exhaled breath analysis by electronic nose in airways 
disease. Established issues and key questions. 
Clin Exp Allergy. 2013 Jul;43(7):705-15. 
16. Gahleitner F, Guallar-Hoyas C, Beardsmore CS, Pandya HC, Thomas CLP. 

 
 
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Metabolomics pilot study to identify volatile organic compound markers of childhood asthma in exhaled 
breath. Bioanalysis. 2013; 5(18): 2239-2247. 
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obstructive pulmonary disease. Thorax 2012 Nov; 67(11):970-6. 15. 
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from community-acquired pneumonia: systematic review and meta-analysis. Thorax. 2010 
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Predicting survival in heart failure: a risk score based on 39 372 patients from 30 studies. 
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20. Steer J, Norman EM, Afolabi OA, Gibson GJ, Bourke SC. Dyspnoea severity and pneumonia as predictors 
of in-hospital mortality and early readmission in acute exacerbations of COPD. Thorax. 2012 Feb;67(2):117-
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21. Roche N, Zureik M, Soussan D, et al. Predictors of outcomes in COPD exacerbation cases presenting to 
the emergency department. Eur Respir J 2008;32:953-61. 
22. Tsimogianni AM, Papiris SA, Stathopoulos GT, et al. Predictors of outcome after exacerbation of chronic 
obstructive pulmonary disease. J Gen Intern Med 2009;24:1043-8. 
23. Coats TJ, Shakur H. Consent in emergency research: new regulations. 
Emerg Med J. 2005 Oct;22(10):683-5. Review.  
24.van der Ploeg T, Austin PC, Steyerberg EW. Modern modelling techniques are data hungry: a simulation 
study for predicting dichotomous endpoints. BMC Med Res Methodol. 2014 Dec 22;14:137. doi: 
10.1186/1471-2288-14-137. 
 
 

 
 
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APPENDIX A: RECIVA breath sampling device 
 

 
 
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20. APPENDIX B: RECIVA CE Marking Authorisation 
 
 
 

 
 
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21. APPENDIX C: Interim analysis and sample size calculation  
 
21.1 EMBER Sample Size Estimates 
An interim analysis was conducted in September 2017 to determine the sample size of the ember 
acute study. 
21.2 Closed formula for logistics regression models 
82 acutely breathless adults (asthma:35, community acquired pneumonia :16, COPD:26) and 17 
healthy controls were utilised for the analysis. 
A panel of 10 pre specified aldehyde biomarkers derived from GCMS analysis were supplied. The 
biomarkers were normalised to a common standard and were not background subtracted. 
A closed formula from Hsieh et al, Statis med 1998, was utilised to calculate sample sizes from 
logistic regression models of the 10 aldehydes with acute breathlessness as the outcome 
measure. The sample size estimates are also relevant to acute class comparions vs. the sum of 
other acute classes. 
The results of the analyses are outlined in the figure below (each BRI number annotates a specific 
aldehyde). 
 
Based upon the sample size estimates we would have an 80% power at the 5 % significance level 
to detect an odds ratio of association of 1.2 for a given disease class with 55 patients per disease 
class. Given the fact that MRC EMBER seeks to discover and replicate breath VOCs amongst 5 
adults classes (CAP, heart failure, COPD, asthma and healthy aged matched subjects).  
We would require 110 patients per class – 550 patients across the program to achieve these aims. 
 
21.3 Closed formula for sensitivity and specificity of disease biomarkers 
Commented [CSB1]: This is not a complete sentence. There 
is a clause missing, or else the punctuation needs adjustment. 

 
 
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The closed formulae in Tilaki-Hajjan et al, Journal of Biomedical Informatics 48(2014), 193-2014, 
were also utilised to understand the power that the samples size above would provide with respect 
to biomarker sensitivity and specificity. 
The following assumptions were made: 
- 
That a sensitivity of 80 % with a precision (d) of between 5-8% would provide a useful 
biomarker capable of ‘ruling out’ an acute class.  The same target was applied to specificity. 
- 
We assume a prevalence of acute breathlessness of 80% as the MRC EMBER campaign 
uses acute breathlessness as the initial stratification tool for recruitment and 1:5 patients 
recruited will be non-breathless healthy controls. We aim to balance group sizes across 
classes equally. 
- 
A type 1 error rate of 0.05 and power of 95% CI level, 90% CI level 
- 
Note for a prevalence ≥ 50% 𝑁𝑠𝑝𝑒𝑐≥𝑁𝑠𝑒𝑛𝑠  is always the case, so Nspec  is the number we 
need to consider. 
 
95% CI, precision ± 5% 
Nsens= 1.962 x 0.8 x 0.2 / 0.052 x 0.8 = 307 
Nspec= 1.962 x 0.8 x 0.2 / 0.052 x 0.2 = 1,230 
 
90% CI, precision ± 5% 
Nsens= 1.642 x 0.8 x 0.2 / 0.052 x 0.8 = 215 
Nspec= 1.642 x 0.8 x 0.2 / 0.052 x 0.2 = 861 
To obtain n near 550, increase the maximum marginal error d (i.e. decrease the precision) 
95% CI, precision ± 8%, Prevalence = 80%, Sensitivity and              Specificity=80 % 
 
Nsens = 120 
Nspec  = 480 
 
21.4 Summary 
550 patients (110 per class: healthy, acute asthma, acute COPD, community acquired pneumonia, 
acute heart failure) will be required for combined discovery and replication to (i) detect a minimum 
odds ratio for association of breath VOCs and acute breathlessness of 1.2 or between acute 
breathlessness class (e.g. pneumonia with remaining categories as a reference).  

 
 
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This sample size and group allocation will give us sufficient power to discover a disease specific 
VOC over a 95% CI with a specificity of 80% ± 8%, sensitivity of 80% ± 8% at an α = 0.05. The 
sample size will give us sufficient power to replicate based upon biomarker sensitivity but not 
specificity. 
The power calculation assume that ALL acute patients have one or more of the following (i) patient 
defined acute breathlessness or (ii) a unit change in eMRC (assuming that they can quantify their 
usual baseline eMRC). 
 
21.5 Recruitment Plan: Acute Adult Study 
Disease Category 
Discovery 
Replication 
Acute Adult Asthma 
55 
55 
Acute COPD 
55 
55 
Acute Heart Failure 
55 
55 
Community Acquired 
Pneumonia 
55 
55 
Adult healthy volunteers 
55 
55 
 
21.6 Paediatric Recruitment Plan: Acute Study 
Acute childhood wheeze and or confirmed asthma. 
Revised to 50 Discovery and 25 replication, based upon current recruitment. 
21.7 Stable Disease Recruitment Plan 
This should be opportunistic and support the following ongoing initiatives 
 
Mologics COPD study 
 
Mepolizumab severe asthma early response study 
 
Sushila Natarajan ACF: project April 2018 (small healthy volunteer repeatabillty study n=12 
healthy adults baseline x2, 2 weeks, 3 months GC-MS ReCIVA, PTRMS (Salman to 
discuss at Exec). 
 
Severe asthma clinical patient samples if possible/samples from aligned BRC studies 
(agreed at the exec) 
Planned acute study recruitment last patient visit 1a 
1st Nov 2018 for combined discovery and replication