Akershus Cardiac Examination (ACE) 4 Study: Pragmatic Randomized Controlled Trial of Early Biomarker Measurements and Structured Feedback in Unselected Patients With Tachypnea
Phase: NA
Conditions: Heart Failure, Tachypnea
Sponsor: University Hospital, Akershus

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ACE 4 Study Protocol Version No.3, December 3, 2023 
    
 
 
 
Akershus Cardiac Examination (ACE) 4 Study: 
Pragmatic randomized controlled trial of early biomarker measurements 
and structured feedback in unselected patients with tachypnea 
 
 
Protocol Identification Number:  ACE 4 Study 
ClinicalTrials.gov Identifier: NCT05699564 
 
 
SPONSOR: 
Akershus University Hospital 
Division of Medicine 
1478 Lørenskog, Norway  
Tel : +47 02900 
 
 
PRINCIPAL INVESTIGATOR: 
Associate Professor Magnus Lyngbakken, 
MD PhD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 93 40 88 37 
E-mail: magnus.lyngbakken@medisin.uio.no 
 
 
 
PROTOCOL VERSION NO. 3 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
CONTACT DETAILS  
Sponsor: 
Akershus University Hospital 
Division of Medicine 
1478 Lørenskog, Norway 
Tel: +47 67960000 
 
Core Study Group 
 
Principal Investigator: 
Associate Professor Magnus Lyngbakken, MD PhD 
Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 93 40 88 37 
E-mail: magnus.lyngbakken@medisin.uio.no 
 
Co-Principal Investigator: 
Professor Helge Røsjø, MD PhD 
Division of Research and Innovation 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway  
Tel:+47 91 54 58 64 
E-mail: helge.rosjo@medisin.uio.no 
  
Investigator: 
 
 
 
Rahul Bhatnagar, MD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 41 21 83 49 
E-mail: rahul.bhatnagar@studmed.uio.no 
  
Investigator: 
Kristian Berge, MD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 41 28 31 28 
E-mail: kristian.berge@gmail.com  
 
Investigator: 
 
 
 
Professor Henrik Schirmer, MD PhD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 992 64 338 
E-mail: henrik.schirmer@medisin.uio.no 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
 
Investigator: 
 
 
 
Torbjørn Wisløff, MSc PhD 
Division of Research and Innovation, 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 928 67 976 
E-mail: torbjorn.wisloff@ahus.no  
 
 
Investigators 
 
Investigator: 
 
 
 
Professor Torbjørn Omland, MD PhD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 401 07 050 
E-mail: torbjorn.omland@medisin.uio.no 
  
 
Investigator: 
Lars Gunnar Klæbo, MD, PhD 
Department of Cardiology, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 975 43 045 
E-mail: lars.gunnar.klabo@ahus.uio.no 
 
Investigator: 
 
 
 
Associate Professor Gunnar Einvik MD, PhD 
Department of Pulmonary Medicine, Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 411 04 542 
E-mail: gunnar.einvik@medisin.uio.no 
  
 
Investigator: 
 
 
 
Professor Stefan James, MD, PhD 
Uppsala Clinical Research Center, 
Uppsala, Sweden 
Tel: +46 70 594 44 04 
E-mail: stefan.james@ucr.uu.se  
 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
SPONSOR SIGNATURE PAGE 
Title: 
Akershus Cardiac Examination (ACE) 4 Study: Pragmatic randomized controlled trial of early 
biomarker measurements and structured feedback in unselected patients with tachypnea   
Protocol ID no: 
ACE 4 Study 
ClinicalTrials.gov 
Identifier: 
NCT05699564 
 
Sponsor signatory approval 
Irene Grundvold 
Chief of Cardiology 
Akershus University Hospital 
 
                  
Signature                          
 
 
 
 
 
       Date 
 
I hereby declare that this Protocol has been developed in compliance with ICH GCP and the applicable 
regulatory requirements: 
Magnus Lyngbakken 
PI 
MD PhD  
Akershus University Hospital  
 
 
Signature 
 
Date 
 
PI signatory approval 
I hereby declare that I will conduct the study in compliance with the Protocol, ICH GCP, and the applicable 
regulatory requirements: 
Name:   
Title:  
 
 
PI signature 
 
Date 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
PROTOCOL SYNOPSIS 
Intervention 
Early biomarker measurements and structured feedback in patient's electronic medical 
records 
Protocol no.  
ACE 4 Study 
Study phase  
4 
Study title 
Akershus Cardiac Examination (ACE) 4 Study: Pragmatic randomized controlled trial of 
early biomarker measurements and structured feedback in unselected patients with 
tachypnea   
Sponsor 
Department of Cardiology, Division of Medicine, Akershus University Hospital, Norway 
Responsible contact 
persons 
Associate Professor Magnus Lyngbakken, MD PhD 
Division of Medicine 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 93 40 88 37 
E-mail: magnus.lyngbakken@medisin.uio.no 
 
and 
 
Professor Helge Røsjø 
Division of Research and Innovation 
Akershus University Hospital 
Sykehusveien 25 
1478 Lørenskog, Norway 
Tel: +47 91 54 58 64 
E-mail: helge.rosjo@medisin.uio.no 
Funding source(s) 
Akershus University Hospital, University of Oslo, South-Eastern Norway Regional Health 
Authority 
Study center 
Akershus University Hospital, Lørenskog 
Planned 
number 
of 
patients 
Maximum 600 patients 
Timelines 
Estimated study start (first patient first visit [FPFV]): Feb 1, 2023 
Estimated recruitment end (last patient first visit [LPFV]): June 28, 2024 
Follow-up period end date (last patient [LP] off study): Dec 31, 2038 
End of study: Sep 1, 2050 
Background and rationale 
Patients hospitalized with tachypnea, defined as respiratory rate ≥20/ min, have substantial 
mortality and may suffer from different conditions, including acute heart failure (HF). 
Symptoms of HF can be difficult to identify and ~15% of patients with HF will not be 
correctly diagnosed by the treating physician in the Emergency Department (ED). 
Biomarkers like B-type natriuretic peptides (BNPs) and cardiac troponins improve 
diagnostic accuracy and risk stratification. Whether early, structured biomarker assessment 
and structured feedback in the patient's electronic health records (EHR) improve 
management and outcomes among unselected patients with tachypnea have previously 
not been explored in a randomized controlled trial. 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
Study objectives  
Primary endpoint  
To determine whether early structured biomarker assessment in unselected patients with 
tachypnea extends the time to the first event for either (1) all-cause readmission or (2) all-
cause mortality; i.e. time to the combined endpoint, compared to the current strategy/ 
standard care  
  
Secondary endpoints 
• 
To estimate the difference in hospital length of stay during the index hospitalization for 
strategy with early structured biomarker assessment compared to current strategy/ 
standard care in unselected patients with tachypnea 
• 
To estimate the difference in length of stay in Intensive Care Unit/Medical Intensive 
Care Unit/ Cardiac Intensive Care Unit during the index hospitalization for strategy with 
early structured biomarker assessment compared to current strategy/ standard care in 
unselected patients with tachypnea  
• 
To estimate the difference in 30-day all-cause readmission for strategy with early 
structured biomarker assessment compared to current strategy/ standard care in 
unselected patients with tachypnea 
• 
To compare time to first all-cause readmission during follow-up for strategy with early 
structured biomarker assessment compared to current strategy/ standard care in 
unselected patients with tachypnea 
• 
To compare total number of all-cause readmissions during follow-up for strategy with 
early structured biomarker assessment compared to current strategy/ standard care in 
unselected patients with tachypnea 
• To estimate the difference in all-cause mortality during follow-up for strategy with early 
structured biomarker assessment compared to current strategy/ standard care in 
unselected patients with tachypnea 
• To estimate the difference in total cost for strategy with early structured biomarker 
assessment compared to current strategy/ standard care in unselected patients with 
tachypnea 
• To estimate the difference in all-cause mortality during the index hospitalization for 
strategy with early structured biomarker assessment compared to current strategy/ 
standard care in unselected patients with tachypnea 
• To estimate the difference in the cardiac troponin T and/or I and B-type natriuretic 
peptide and/or N-terminal pro-B-type natriuretic peptide concentrations from hospital 
admission to discharge for strategy with early structured biomarker assessment 
compared to current strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in guideline-defined medical therapy for HF, as defined by 
international guidelines, at discharge for strategy with early structured biomarker 
assessment compared to current strategy/ standard care in patients adjudicated with 
HF diagnosis and categorized into HF with reduced or mildly reduced ejection fraction 
(HFrEF/ HFmrEF) and HF with preserved ejection fraction (HFpEF) 
• 
To estimate cost-utility for the strategy with early structured biomarker assessment 
compared to current strategy/ standard care in unselected patients with tachypnea 
 
Total follow-up is permitted until 2038. We will assess the primary endpoint after 12 month 
follow-up from randomization; i.e. patients are followed-up either for 12 months or to the 
time of death (if this happens before 12 months from randomization). We will also report 
data for extended follow-up after 3 years, 5 years, and 10 years for the primary outcome.  

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ACE 4 Study Protocol Version No.3, December 3, 2023 
 
For secondary endpoints we will report data after the index hospitalization, after 30 days 
follow-up, after 1 year, 3 years, 5 years, and 10 years. Cost-utility will be evaluated based 
on data reported up to and including 12 months, and updated after 5 years. 
 
Explorative endpoints: 
• 
Assessing primary and secondary outcomes in the subgroup of patients classified as 
hospitalized due to HF, as assessed by the adjudication committee  
• 
Assessing primary and secondary outcomes in HF patients, classified by the 
adjudication committee, with patients stratified by LVEF 50%; e.g. HFrEF/ HFmrEF vs. 
HFpEF 
• 
Assessing primary and secondary outcomes with patients stratified by biomarker 
concentrations measured on the first study visit (study inclusion), including B-type 
natriuretic peptides, cardiac troponins, and additional biomarkers 
• 
Assessing accuracy for clinical and biochemical diagnostic scores for diagnosing 
HFpEF; e.g. the H2FPEF and HFA-PEFF scores 
• 
The ACE 4 Study will phenotype participants with short-term Holter recordings (≤24 h), 
transthoracic echocardiography (TTE), and biospecimens collection on admission, 
discharge, and after 6 months for later molecular characterization in all participants 
and we will compare phenotypic traits with adjudicated diagnosis and clinical outcomes 
during follow-up 
 
Study design  
Single-center, pragmatic randomized controlled trial of a non-pharmacological intervention   
Screening phase: <24 h of admission to ED.  
Study phase: All patients will be randomized <24 h of admission to ED 
 
Total follow-up is permitted until 2038. We will assess the primary endpoint after 12 month 
follow-up from randomization; i.e. patients are followed-up either for 12 months or to the 
time of death (if this happens before 12 months from randomization). We will also report 
data for extended follow-up after 3 years, 5 years, and 10 years for the primary outcome.  
 
For secondary endpoints we will report data after the index hospitalization, after 30 days 
follow-up, after 1 year, 3 years, 5 years, and 10 years.  
Patient population 
Inclusion 
• 
Patients ≥18 years old 
• 
Tachypnea defined as respiratory rate ≥20/min, as noted in the National Early 
Warning Score (NEWS) 2 classification performed on admission in the Emergency 
Department and reported in the patient' EHR, which is performed as clinical 
routine for all admission at Akershus University Hospital  
• 
Admission to Departments under the Division of Medicine at Akershus University 
Hospital, except the Department of Neurology 
• 
<24 h from hospital admittance to inclusion in the study  
• 
Signed written informed consent during the initial phase of the hospitalization 
 
Exclusion  
• 
Previously included into the study (in case of patients presenting with a second 
hospitalization during the study period) 
• 
Known or suspected cancer outside of local control, documented in medical 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
records, at the time of patient inclusion or diagnosed in relation to the index 
hospitalization   
• 
Neurological condition with short life expectancy; e.g. ALS, documented in 
medical records during screening prior to study entry 
• 
Other non-cardiac disease with life expectancy below 1 year, documented in 
medical records during screening prior to study entry   
• 
Obvious non-cardiac cause for tachypnea based on medical records and clinical 
findings during screening prior to study entry; e.g. anaphylaxis in young patient 
with known allergy, dyspnea after direct chest trauma, or young patient with fever 
and positive Covid-19 test on admission. 
• 
Patient assessed as non-Internal Medicine patient; e.g. surgical patient 
• 
Patients unwilling or unable to comply with the protocol, including Glasgow Coma 
Scale <13 on the time of study inclusion 
• 
Patients that are intubated for invasive ventilatory therapy before or shortly after 
hospital admission 
• 
History of non-compliance to medical management and patients who are 
considered potentially unreliable, based on documentation in medical records, 
during screening prior to study entry 
• 
History or evidence of alcohol or drug abuse with the last 12 months, based on 
medical records and clinical findings during screening prior to study entry, that will 
influence study participation  
• 
Any surgical or medical condition, based on medical records and clinical findings 
during screening prior to study entry, that will impair the ability of the patient to 
participate in the study 
Visit 
schedule 
and 
assessments 
Early biomarker assessment consists of the following examinations: the cardiac biomarkers 
N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin 
T (hs-cTnT), which will be recorded in all patients on hospital admission as part of routine 
care. In the group randomized to early biomarker assessment, we will use a priori-decided 
cutoffs and a specific biomarker algorithm to characterize patients as rule out, 
undetermined ("grey zone"), and rule in for HF. In the control group, biomarker 
concentrations will be provided in the electronic laboratory report without a specific note in 
the text-based section of the patient' EHR, which is the current standard for reporting 
laboratory data today at Akershus University Hospital.  
 
The patient will need to have signed a written informed consent prior to any study-specific 
note being included in the patient' EHR. In the group randomized to early biomarker 
assessment and structured feedback, biomarker-based assessment concerning probability 
for HF will be entered into the EHR of the individual patient.  
 
We will not provide any recommendations relating to diagnostic work up for individual 
patients. The study participants will receive all examinations considered appropriate by the 
treating physician, which includes TTE in the control group. Hence, there will be no 
restrictions to the diagnostic testing of our study participants, regardless of randomization 
status. If requested by the treating physician, a full TTE will be performed by trained study 
personnel in both groups. The TTE protocol will assess atrial and ventricular dimensions, 
systolic and diastolic properties of the left ventricle, right ventricular systolic function, the 
cardiac valves, pericardial effusion, and estimate arterial pulmonary pressure + look for 
other signs indicative of pulmonary hypertension. The conclusion of the TTE will be 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
recorded in the patient' EHR, according to standard procedure. The decision of the patient 
to participate or to decline participation into the study will not influence the treatment that is 
offered to the patient at Akershus University Hospital and all patients, regardless of study 
participation, will receive current strategy/ standard care treatment. 
 
We will not provide any treatment recommendation for individual patients. However, we will 
include a short paragraph related to information for pharmacological treatment of patients 
with HFrEF (LVEF≤50%), HFmrEF (LVEF 40-50%), and HF with recovered LVEF 
according to the European Society of Cardiology (ESC) Heart Failure Guidelines 2021, 
which is the most recent recommendations for HF patients from the ESC. For such patients 
we will inform the physician that β-blocker, SGLT2-inhibitor, inhibition of renin-angiotensin 
with ACE inhibitors or angiotensin-neprilysin inhibition, and aldosterone inhibition are 
recommended. We will also inform that loop diuretics are recommended for volume control/ 
decongestion in all patients with HF according to the ESC guidelines. The recent ESC 
guidelines have no clear pharmacological recommendations for patients with LVEF≥50% 
(HFpEF), but based on emerging data from randomized clinical trials demonstrating effects 
from SGLT2-inhibitors across the spectrum of LVEF in HF patients (e.g. DELIVER trial) we 
will inform the treating physician about SGLT-2-inhibitiors in HFpEF patients. We will also 
inform the treating physician about aldosterone inhibition, as aldosterone inhibition reduced 
HF hospitalizations in the TOPCAT trial (secondary endpoint) and reduced the primary 
endpoint of TOPCAT in post-hoc analysis. Given that treatment of HFpEF seems to be 
developing with more documentation related to effect by pharmacological therapy on HF 
hospitalizations and mortality, we will update the information to the treating physicians and 
include new, ESC-reported recommendations if released during the study inclusion period. 
The recent US (AHA/ACC/HFSA) guidelines for heart failure (2022) supports this strategy.   
 
We will also inform that, according to several ESC guidelines, patients with suspected 
acute coronary syndromes are recommended to be clinically assessed for coronary artery 
disease and treated according to current guidelines. We will also, on a general basis, 
inform the treating physician that patients with atrial fibrillation should receive guideline-
recommended treatment, including anti-coagulation and frequency control.  
 
We apply for permission to establish a real-time electronic surveillance of all patients 
hospitalized at the Division of Medicine, Akershus University Hospital that have tachypnea 
as defined by NEWS2 scoring, which is part of clinical routine. Only dedicated study 
personnel will have access to this information, which will be presented to the study team as 
a list that is stored on secure servers. We also apply for permission to check in the EHR of 
patients with tachypnea whether the patients fulfill inclusion criteria and do not meet 
exclusion criteria of the ACE 4 Study. After the study team has identified eligible patients, 
the study team will obtain written, informed consent from the patient. After this, trained 
study personnel will collect additional information on current and prior medical history by 
the strategy previously successfully used in the ACE 2 Study. This information will be 
checked against the EHR of the patient. All participants of the study, regardless of 
randomization status, will be subjected to blood sampling for biobank (admission and 
discharge), short-term ECG monitoring (≤24 h), as well as TTE if this has not yet been 
performed, after clinical stabilization and prior to hospital discharge. The results of short-
term ECG monitoring is for research-use only purposes and can be evaluated also after 
patient discharge. Hence, the results for the short-term ECG monitoring will not be 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
communicated back to the patient or the treating physician. Analogously; the discharge 
echocardiography is for research-use only purposes and will not be communicated back to 
the treating physician or patient. This discharge echocardiography will be performed only in 
the situation that no echocardiography was ordered by the treating physician during the 
index hospitalization. Still, if research personnel identify unrecognized, gross pathology on 
online analysis while performing the examination, like left ventricular ejection fraction<40%, 
right ventricular strain indicative of possible pulmonary embolus, or pericardial effusion with 
risk of cardiac tamponade, this information will be communicated directly to the treating 
physician and also noted in the patient' EHR. All echocardiographic examinations will later 
also be examined by off line-analysis (after the patient has been discharged), and these 
examinations are for research-use only and results will not be communicated back to the 
patient.  
 
To ensure that the selected population for the ACE 4 Study is representative to all patients 
hospitalized with tachypnea during the same period, we ask permission to collected basic, 
anonymized information related to the patients eligible for inclusion, but not included into 
the study (e.g. not enough resources to include all patients on a given day). For these 
patients we will only collect the basic information age, sex, and hospital length of stay for 
the index hospitalization, and we will anonymize the data before storage (i.e. not possible 
to correlate data to individual patients).  
  
The index hospitalization for all included patients will be classified by an endpoint 
committee of two expert physicians working independently, as (1) HF, (2) non-HF, but with 
myocardial dysfunction, and (3) non-HF and normal LV function. This process and these 
categories are analogous to the system previously used in the ACE 2 Study and 
international studies for adjudication committees in such studies.  
 
We will invite included patients back for a 6-month outpatient visit to register medication 
and to collect biospecimen for biobank storage. The rationale for biospecimen collection 
also after 6 months is to assess serial concentration of biomarker over time, and to 
correlate changes in biomarker concentration with medication. This 6-month visit is detailed 
in the signed informed consent for this study, where patients are informed that we can 
“contact the patient after 1, 3, and 6 months, 1 year, 2 years, 4 years, 6 years, 8 years, 10 
years, 12 years, and 15 years, and also other time points”. 
Data 
management 
and 
statistical analysis  
This is a single-center, pragmatic randomized controlled trial of early structured biomarker 
assessment in patients hospitalized to Internal Medicine with tachypnea, defined as 
respiratory rate ≥20/ min, as noted in the NEWS 2 classification performed on admission in 
the Emergency Department and reported in the patient' EHR.  
 
The data will be summarized with respect to demographic and baseline characteristics, 
efficacy observations and measurements, and safety observations and measurements. 
Categorical data will be presented as absolute frequencies and percentages. For 
continuous data, N, mean±SD or median (quartile 1-3) will be presented. Time to event 
variables and Kaplan-Meier product-limit estimates will be presented stratified by 
intervention group. The primary safety and efficacy analyses will be conducted on all 
patient data at the time of inclusion of the last patient. The final diagnosis of the 
hospitalization will be established by an adjudication committee with two senior physicians 
reviewing all information available on the patients, including information on the clinical 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
outcome of the patient within 90 days after discharge from the index hospitalization. 
Financial resource utilization will be performed by collecting information on hospital length 
of stay and type of hospital ward (ICU/CCU vs. standard), by collecting information on all 
supplementary tests performed during or in conjunction with the index hospitalization, and 
data from local, regional and national registries. 
   
The primary end-point is whether early structured biomarker assessment in unselected 
patients with tachypnea extends the time to the first event for either (1) all-cause 
readmission or (2) all-cause mortality; i.e. time to the combined endpoint, compared to the 
current strategy/ standard care. We will assess the primary endpoint after 12 month follow-
up from randomization; i.e. patients are followed-up either for 12 months or to the time of 
death (if this happens before 12 months from randomization). We will also report data for 
extended follow-up after 3 years, 5 years, and 10 years for the primary outcome.  
Secondary endpoints relate to all-cause mortality, hospital length of stay, need for ward in 
ICU/CCU, total number of re-admissions, resource utilization, medical therapy, and 
changes in biomarker concentrations. For secondary endpoints we will report data after the 
index hospitalization, after 30 days follow-up, after 1 year, 3 years, 5 years, and 10 years. 
We will include a Data Safety and Monitoring Board (DSMB), which will meet after 100, 
200, and 400 included patients. The DSMB will assess key data like primary and some 
secondary endpoints in the two groups. The DSMB may then recommend that the study 
continues or stops based on safety or futility.  
 
The ACE 4 Study will have a large prospective epidemiological section related to various 
types of biomarkers in the total cohort or different subgroups and also to additional 
molecular biological characterizations. The planned sub-studies relate to the performance 
of novel cardiovascular biomarkers for diagnostic and prognostic accuracy compared to 
clinical assessment, established biomarkers, and individual echocardiographic indices. We 
will also examine ECG variables and echocardiographic parameters and link these 
variables to diagnosis during the index hospitalization and clinical outcomes.  
 
Patient follow-up and adjudication of clinical events during follow-up will be performed by 
either linking our data to national registries or by reviewing patient medical records or 
records from other health care providers. We will contact the patients for clinical information 
(e.g. events) during follow-up and for a physical visit after 6 months. With biospecimen 
collection also after 6 months, we plan to assess serial changes in biomarker concentration 
and examine correlation between biomarker concentration and medication. We will also 
investigate associations between biomarker concentration after 6 months, and between 
change in biomarker concentration from inclusion to 6 month, and clinical outcomes during 
follow-up. In studies of biomarkers and prognosis, the patients may also be stratified into 
subgroups based on the adjudicated diagnosis of the index hospitalization (e.g. acute heart 
failure, acute exacerbation of chronic obstructive pulmonary disease, bacterial pulmonary 
infection, etc.) and we will use multivariable statistical models to assess the individual 
performance of biomarkers/other tests.    
 
A health economic evaluation will be performed after completion of the primary endpoint 
analyses at 12 months. The health economic evaluation will be evaluating costs per quality-
adjusted life years, i.e. a cost-utility analysis. 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
Statistical power calculation is based on data from the ACE 2 Study, which included 314 
patients with similar clinical characteristics as expected for the ACE 4 Study cohort. The 
power calculation is based on the assumption that approximately 45% of patients 
hospitalized with tachypnea will be diagnosed with acute HF and that a substantial 
proportion of the patients with non-HF related dyspnea will exhibit evidence of myocardial 
dysfunction; in total 65% of the ACE 2 cohort was classified as hospitalized with myocardial 
dysfunction. The 1-year readmission/all-cause mortality rate in the ACE 2 Study was 
approximately 40%, and we expect a rate reduction to 25% in the group with early 
structured biomarker assessment among patients adjudicated to have myocardial 
dysfunction. Based on these numbers, we will need to randomize 152 patients to early 
structured biomarker assessment and 152 patients to the control group to have 80% 
probability to detect a significant difference (p<0.05) between the two groups. Based on 
data from the ACE 2 Study, 65% of the population will suffer from myocardial dysfunction, 
thus we will need to randomize 234 patients to early cardiological assessment. With a 1:1 
randomization strategy taking into account possible patient dropouts, we will aim to include 
at least 500 patients in the ACE 4 Study. However, we ask permission to include up to 600 
patients if additional pilot experiments prior to study commencement indicate a need for a 
larger sample size. 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
TABLE OF CONTENTS 
CONTACT DETAILS .......................................................................................................................................................... 2 
SPONSOR SIGNATURE PAGE ......................................................................................................................................... 4 
PROTOCOL SYNOPSIS .................................................................................................................................................... 5 
TABLE OF CONTENTS ................................................................................................................................................... 13 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ......................................................................................... 15 
1 
INTRODUCTION ...................................................................................................................................................... 17 
1.1 
Background – Diagnostic Challenges in Patients with Acute Dyspnea .............................................................. 17 
1.2 
Background –Testing in Patients with Acute Dyspnea and Tachypnea ............................................................. 17 
1.3 
Summary of Previous Clinical Studies and Current Strategies .......................................................................... 19 
1.3.1 Diagnostic Accuracy in Patients with Acute Dyspnea and Tachypnea.............................................................. 19 
1.3.2 Current Strategies in Patients with Acute Dyspnea and Tachypnea ................................................................. 19 
1.4 
Rationale for the Study ....................................................................................................................................... 19 
2 
STUDY OBJECTIVES ............................................................................................................................................. 20 
2.1 
Endpoints ........................................................................................................................................................... 20 
3 
STUDY POPULATION ............................................................................................................................................. 21 
3.1 
Selection of Study Population ............................................................................................................................ 21 
3.2 
Inclusion Criteria ................................................................................................................................................ 22 
3.3 
Exclusion Criteria ............................................................................................................................................... 23 
3.4 
Patient registration ............................................................................................................................................. 23 
4 
STUDY EXECUTION ............................................................................................................................................... 23 
4.1 
Patient Screening, Inclusion, and Randomization .............................................................................................. 23 
4.2 
Early Biomarker-based Cardiological Assessment ............................................................................................ 24 
4.3 
Patient Follow-Up During the Index Hospitalization and Data Storage .............................................................. 25 
4.4 
Adjudication of Diagnosis and Clinical Events ................................................................................................... 26 
4.5 
Quality of Life and Resource Utilization ............................................................................................................. 27 
4.6 
Deep Phenotyping and Exploratory Endpoints ................................................................................................... 27 
5 
DATA MANAGEMENT AND STATISTICAL ANALYSIS ........................................................................................ 29 
5.1 
Electronic Case Report Forms (eCRFs) ............................................................................................................. 29 
5.2 
Source Data ....................................................................................................................................................... 29 
5.3 
Source Data Verification .................................................................................................................................... 29 
5.4 
Storage of Study Documentation ....................................................................................................................... 30 
5.5 
Study Design ...................................................................................................................................................... 30 
5.6 
Sample Size & Statistical Considerations .......................................................................................................... 30 
5.7 
Safety Analysis ................................................................................................................................................... 30 
5.8 
Interim Analysis .................................................................................................................................................. 31 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
6 
ETHICAL AND REGULATORY REQUIREMENTS ................................................................................................. 31 
6.1 
Ethical Considerations ....................................................................................................................................... 31 
6.1.2 Informed Consent.............................................................................................................................................. 31 
6.2 
Obligations of Investigators ................................................................................................................................ 32 
6.3 
Record Retention ............................................................................................................................................... 32 
6.4 
Audits ................................................................................................................................................................. 32 
6.5 
Publication Policy ............................................................................................................................................... 33 
7 
STUDY MANAGEMENT .......................................................................................................................................... 33 
7.1 
Investigator Delegation Procedure ..................................................................................................................... 33 
7.2 
Study Amendments ............................................................................................................................................ 33 
7.3 
Audit and Inspections ......................................................................................................................................... 33 
8 
TRIAL SPONSORSHIP AND FINANCING .............................................................................................................. 33 
9 
TRIAL INSURANCE ................................................................................................................................................ 33 
10 ACKNOWLEDGEMENTS ........................................................................................................................................ 33 
11 APPENDIX ............................................................................................................................................................... 35 
11.1 Echocardiographic protocol ................................................................................................................................ 35 
12 REFERENCES ......................................................................................................................................................... 36 
11  APPENDIX…………………………………………………………………………………………………………………….28
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
Abbreviation or special term 
Explanation 
ACE 2 Study 
Akershus Cardiac Examination 2 Study 
ACE 4 Study 
Akershus Cardiac Examination 4 Study 
ACS 
Acute coronary syndrome 
AECOPD 
Acute exacerbation of chronic obstructive pulmonary disease 
AF 
Atrial fibrillation 
ALS 
Amyotrophic lateral sclerosis 
AMI 
Acute Myocardial Infarction 
AUC 
Area under the curve 
BNP/NT-proBNP 
B-type natriuretic peptide/N-terminal pro-B-type natriuretic peptide 
CRF 
Case Report Forms 
CT 
Computer tomography 
CV/CVD 
Cardiovascular/ cardiovascular disease 
DSMB 
Data Safety and Monitoring Board 
ECG 
Electrocardiogram 
EMR 
Electronic medical records 
ED 
Emergency Department 
FEV1 
Forced expiratory volume in 1 sec 
FVC 
Forced vital capacity 
GOLD 
Global initiative of chronic Obstructive Lung Disease 
HF 
Heart failure 
HFpEF 
Heart failure with preserved ejection fraction 
HFrEF 
Heart failure with reduced ejection fraction 
HR 
Heart rate 
hs-cTnT 
High-sensitivity cardiac troponin T 
JVP 
Jugular venous pulse 
LoS 
Hospital length of stay 
LV 
Left ventricular 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
LVEDD 
Left ventricular end-diastolic dimension 
NEWS2 
National Early Warning Score 2 
NYHA  
New York Heart Association 
PE 
Pulmonary embolus 
PI 
Principal Investigator 
RCT 
Randomized controlled trial 
TTE 
Transthoracic echocardiogram 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
1 
INTRODUCTION 
1.1 Background – Diagnostic Challenges in Patients with Acute Dyspnea  
Patients with dyspnea constitute a heterogeneous group of patients with substantial short- and long-term mortality. 
Major patient groups admitted to the Emergency department (ED) with dyspnea are patients with acute heart failure 
(HF), acute exacerbations of chronic obstructive pulmonary disease (AECOPD), and pulmonary infections. Other 
conditions that can also lead to dyspnea are pulmonary embolus (PE) and asthma. These are all potential life-
threatening conditions and delayed diagnosis are associated with increased morbidity and mortality. In addition, missed 
diagnosis in the ED causes a delay in the initiation of appropriate therapy, which will increase hospital length of stay 
(LoS) and expenditure without improving patient care. An important clinical sign in patients with dyspnea are increased 
breathing rate (tachypnea) and 192 of 314 patients (61%) hospitalized with acute dyspnea and included into the 
Akershus Cardiac Examination 2 (ACE 2) Study, conducted at Akershus University Hospital during 2009-2010, 
demonstrated respiratory rate >20/ min on ED admission, which is characterized as tachypnea.  
Unfortunately, making the correct diagnosis in the ED can be challenging as symptoms overlap between the different 
conditions and clinical signs may not accurately reflect the underlying pathology. Even patients with severe HF, as 
documented by invasive measurements of pulmonary artery pressures, can present without signs considered sensitive 
for acute HF; e.g. increased jugular venous pulse (JVP), pulmonary crackles, and peripheral edemas. Previous reports 
from multicenter trials have found physicians working in the ED to diagnose acute HF with 85% accuracy, as assessed 
by receiver operating statistics (area under the curve [AUC≈0.85]). In line with this, we recently found AUC=0.86 for 
physicians working in the ED at Akershus University Hospital to diagnose acute HF in the ACE 2 Study. Hence, 
diagnostic accuracy for HF among unselected patients with acute dyspnea and tachypnea can be improved. 
1.2 Background –Testing in Patients with Acute Dyspnea and Tachypnea 
Acute HF is a syndrome that is caused by different underlying pathophysiology (Figure 1). Hence, early cardiological 
assessment should cover all processes that potentially could cause acute HF. Current guidelines recommend the use 
of supplementary tests in the ED in patients with suspected HF, including electrocardiogram (ECG), measurements of 
cardiac biomarkers, chest X-ray and early transthoracic echocardiography (TTE). To the best of our knowledge, no 
study has previously tested whether a strategy combining (1) early structured biomarker assessment (a priori-based 
cutoffs) and (2) subsequent standardized reporting in the patient's electronic health records (EHR) will improve clinical 
outcomes compared to biomarker testing and assessment performed by attending physicians (standard care). We will 
base cardiological assessment on a few robust parameters that can be ascertained in all patients and evaluated also by 
junior physicians. 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
ECG 
is 
an 
easily 
available 
supplementary test that is helpful to 
diagnose myocardial ischemia and 
supraventricular 
and 
ventricular 
arrhythmias. ECG assessment may also 
enable the physician to suspect prior 
myocardial injury or structural heart 
disease, which then should be further 
explored with additional tests, including 
TTE. The importance of obtaining 
information from the ECG can also be 
appreciated by the association between 
atrial fibrillation (AF) and acute HF. AF 
and other supraventricular tachycardias 
are especially associated with acute HF 
in patients with HFpEF, as these patients 
have impaired left ventricular (LV) 
relaxation. Hence, HFpEF patients are 
especially vulnerable to tachycardia, 
which will increase left atrial filling 
pressures, increase pulmonary artery 
pressures, 
and 
induce 
pulmonary 
congestion. Unfortunately, high quality echocardiographic images can be difficult to obtain in patients with rapid AF and 
subsequent tachypnea, which underlines the need to base early cardiological assessment on supplementary tests 
besides TTE alone. ECG is also helpful to diagnose acute coronary syndrome in the subgroup of patients with 
tachypnea due to acute coronary artery disease. However, as moderate/minimal ST-T segment abnormalities can be 
caused by tachycardia and tachypnea directly, and as physician experience influences ECG interpretation, we have not 
included ECG in the structured cardiological assessment to be tested in the Akershus Cardiac Examination 4 (ACE 4) 
Study. Moreover, as high-sensitivity cardiac troponin assays will detect even minute myocardial injury, cardiac 
biomarker testing will duplicate and add to the information that are provided by ECG readings. Still, one could argue 
that detection of AF could be included into early cardiology assessment, but given the large overlap in AF prevalence 
between patients with acute HF and non-HF related dyspnea, reflected in a modest AUC=0.65 for AF in the ACE 2 
Study (vs. AUC=0.85 for NT-proBNP), ECG variables seem to have limited additional value to cardiac biomarkers when 
assessing risk of HF. 
Current guidelines recommend early testing of concentrations of B-type natriuretic peptide/N-terminal pro-B-type 
natriuretic peptide (BNP or NT-proBNP) in patients with suspected HF as they provide excellent discrimination between 
HF and non-HF dyspnea and improve physician accuracy for diagnosing HF. Still, BNP/NT-proBNP measurements will 
fail to identify HF in 10-15% of patients admitted with acute dyspnea. Moreover, cutoffs for BNP/NT-proBNP are 
different in patients with HFrEF and HFpEF. Thus, although useful for diagnosing HF, BNP/NT-proBNP concentrations 
should be interpreted in the context of HFrEF/HFpEF. The need to incorporate biomarker concentrations with additional 
knowledge could explain why some studies have found early natriuretic peptide testing to improve patient management, 
while results have not been as clear in other studies. Based on the literature, and also including information from our 
own ACE 2 Study, we will establish a combined NT-proBNP and high-sensitivity cardiac troponin T (hs-cTnT)-algorithm 
that should provide sensitive and specific for HF across the spectrum of HF. The definition of acute myocardial 
infarction also incorporates cardiac troponin testing as an essential criterion for the diagnosis and our algorithm will be 
accurate also to diagnose acute myocardial infarction in unselected patients with tachypnea. We have demonstrated 
that high-sensitivity cardiac troponin testing complements BNP/ NT-proBNP measurements for HFpEF diagnosis in the 
 
Fig.1. Acute HF may be caused by hypertensive heart disease, heart failure with 
preserved ejection fraction (HFpEF), acute coronary syndrome, de novo acute HF, 
or acute-on-chronic HF. The severity of acute HF may differ between New York 
Heart Association (NYHA) functional class II-IV with the most severe cases being 
classified as pulmonary edema (as diagnosed by chest x-rays). From McDonagh 
TA, et al. Eur J Heart Fail 2011;13:1253-60 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
ACE 2 Study, and therefore will combine hs-cTnT and BNP/ NT-proBNP to diagnose HF in unselected patients with 
tachypnea in the ACE 4 Study.    
TTE is a non-invasive method to determine myocardial structure and function and is recommended as early as possible 
in patients with suspected acute HF. Although easy to perform by trained personnel, TTE is greatly influenced by the 
experience of the examiner and inter- and intrarater variability can be profound. In patients with tachypnea image 
quality will be suboptimal as the patient cannot lie down and there are rapid, high-frequency movements of the chest 
during respiratory work. In theory, performing early echocardiography in all patients with tachypnea could therefore 
impair patient safety and increase the time to appropriate therapy, as physicians will be preoccupied with performing 
echocardiography and potentially also could focus too strongly on HF as the cause of tachypnea. In this scenario, early 
TTE may divert the attention from other important etiologies that also can cause tachypnea, including AECOPD, PE, 
and pulmonary infections. Hence, we believe a standardized protocol based on the easily available cardiac biomarkers 
BNP/ NT-proBNP and high-sensitivity cardiac troponin, followed by structured feedback in the patient's EMR, will 
provide a balanced strategy to increase diagnostic accuracy for HF among unselected patients with tachypnea. Still, 
elevated biomarker concentrations will need additional work up, including TTE, and therefore we expect that biomarker 
profiles indicative of HF and structured feedback in the patient' EMR will result in referral by the treating physician for 
early TTE in the majority of cases.  
1.3 Summary of Previous Clinical Studies and Current Strategies 
1.3.1 Diagnostic Accuracy in Patients with Acute Dyspnea and Tachypnea 
In general, physician AUCs for diagnosing HF in patients hospitalized with acute dyspnea (and thereby also 
tachypnoea) have been reported to be in the range of 0.76-0.90, and the heterogenous result could relate both to 
differences in the population assessed and the quality of diagnostic work up at the different centers. Previous 
multicenter trials have reported AUCs of 0.85 and 0.86 and we have found AUC=0.86 for physicians working in the ED 
of Akershus University Hospital to diagnose HF in patients with acute dyspnea (ACE 2 Study). Accordingly, novel 
strategies are needed to improve diagnostic accuracy for HF in patients hospitalized with dyspnea and tachypnea.  
1.3.2 Current Strategies in Patients with Acute Dyspnea and Tachypnea 
The current strategy for attending physicians in patients with acute dyspnea and suspected HF is to obtain patient 
history and perform clinical examination before ordering supplementary tests like ECG recordings, BNP/NT-proBNP 
measurements, chest X-ray and high-sensitivity cardiac troponin testing in the majority of cases. Normally, TTE is 
performed in the acute/subacute phase.     
1.4 Rationale for the Study 
Acute HF constitutes approximately 40% of patients hospitalized with acute dyspnea (and therefore tachypnea). In 
addition, approximately 1/3 of patients hospitalized with non-HF related dyspnea will exhibit evidence of myocardial 
dysfunction. Accordingly, the total proportion of patients hospitalized with acute dyspnea and tachypnea with evidence 
of myocardial injury or dysfunction will be 60-65%. Given that physician accuracy for diagnosing HF in the ED is 
AUC=0.85, strategies that increase diagnostic accuracy should improve patient care. Hence, in the ACE 4 Study we will 
test the strategy whether early biomarker assessment and structured feedback in the patient' EMR reduces clinical 
endpoints in unselected patients with tachypnea.  

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ACE 4 Study Protocol Version No.3, December 3, 2023 
2 
STUDY OBJECTIVES 
The primary endpoint of the study will be whether early biomarker assessment and structured feedback in the patient' 
EHR assessment in unselected patients with tachypnea extends the time to the first event for either (1) all-cause 
readmission or (2) all-cause mortality; i.e. time to the combined endpoint, compared to the current strategy/ standard 
care. We hypothesize that the strategy of early biomarker assessment and structured feedback in unselected patients 
with tachypnea will also affect endpoints related to morbidity and health economics, and also that the strategy will help 
decipher underlying pathophysiology in acute HF, including conditions such as acute coronary syndromes, HFpEF, and 
HFrEF. We will also perform prespecified stratified analyses according to HF diagnosis and HF phenotype.  
The study participants will be extensively characterized with echocardiography, ECG monitoring, and detailed 
molecular characterization and there will be explorative endpoints related to also these analyses.   
2.1 Endpoints 
 
Primary endpoint  
To determine whether early structured biomarker assessment in unselected patients with tachypnea extends the time to 
the first event for either (1) all-cause readmission or (2) all-cause mortality; i.e. time to the combined endpoint, 
compared to the current strategy/ standard care 
 
Secondary endpoints 
• 
To estimate the difference in hospital length of stay during the index hospitalization for strategy with early 
structured biomarker assessment compared to current strategy/ standard care in unselected patients with 
tachypnea 
• 
To estimate the difference in length of stay in Intensive Care Unit/Medical Intensive Care Unit/ Cardiac Intensive 
Care Unit during the index hospitalization for strategy with early structured biomarker assessment compared to 
current strategy/ standard care in unselected patients with tachypnea  
• 
To estimate the difference in 30-day all-cause readmission for strategy with early structured biomarker assessment 
compared to current strategy/ standard care in unselected patients with tachypnea 
• 
To compare time to first all-cause readmission during follow-up for strategy with early structured biomarker 
assessment compared to current strategy/ standard care in unselected patients with tachypnea 
• 
To compare total number of all-cause readmissions during follow-up for strategy with early structured biomarker 
assessment compared to current strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in all-cause mortality during follow-up for strategy with early structured biomarker 
assessment compared to current strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in total cost for strategy with early structured biomarker assessment compared to current 
strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in all-cause mortality during the index hospitalization for strategy with early structured 
biomarker assessment compared to current strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in the cardiac troponin T and/or I and B-type natriuretic peptide and/or N-terminal pro-B-
type natriuretic peptide concentrations from hospital admission to discharge for strategy with early structured 
biomarker assessment compared to current strategy/ standard care in unselected patients with tachypnea 
• To estimate the difference in guideline-defined medical therapy for HF, as defined by international guidelines, at 
discharge for strategy with early structured biomarker assessment compared to current strategy/ standard care in 
patients adjudicated with HF diagnosis and categorized into HF with reduced or mildly reduced ejection fraction 
(HFrEF/ HFmrEF) and HF with preserved ejection fraction (HFpEF) 
• 
To estimate cost-utility for the strategy with early structured biomarker assessment compared to current strategy/ 
standard care in unselected patients with tachypnea 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
 
Total follow-up is permitted until 2038. We will assess the primary endpoint after 12 month follow-up from 
randomization; i.e. patients are followed-up either for 12 months or to the time of death (if this happens before 12 
months from randomization). We will also report data for extended follow-up after 3 years, 5 years, and 10 years for the 
primary outcome.  
 
For secondary endpoints we will report data after the index hospitalization, after 30 days follow-up, after 1 year, 3 
years, 5 years, and 10 years. Cost-utility will be evaluated based on data reported up to and including 12 months, and 
updated after 5 years 
 
Explorative endpoints: 
• 
Assessing primary and secondary outcomes in the subgroup of patients classified as hospitalized due to HF, as 
assessed by the adjudication committee  
• 
Assessing primary and secondary outcomes in HF patients, classified by the adjudication committee, with patients 
stratified by LVEF 50%; e.g. HFrEF/ HFmrEF vs. HFpEF 
• 
Assessing primary and secondary outcomes with patients stratified by biomarker concentrations measured on the 
first study visit (study inclusion), including B-type natriuretic peptides, cardiac troponins, and additional biomarkers 
• 
Assessing accuracy for clinical and biochemical diagnostic scores for diagnosing HFpEF; e.g. the H2FPEF and 
HFA-PEFF scores 
• 
The ACE 4 Study will phenotype participants with short-term Holter recordings (≤24 h), transthoracic 
echocardiography (TTE), and biospecimens collection during the index hospitalization and after 6 months for later 
molecular characterization in all participants and we will compare phenotypic traits with adjudicated diagnosis and 
clinical outcomes during follow-up 
 
3 
STUDY POPULATION 
3.1 Selection of Study Population 
Over a period from February 1, 2023 until June 28, 2024, the ACE 4 Study will enroll a minimum of 400 patients and a 
maximum of 600 patients hospitalized at Akershus University Hospital with tachypnea. Hence, stopping criterion for the 
trial is either inclusion of 600 patients or June 18, 2024. At Akershus University Hospital, all patients are scored 
according to National Early Warning score 2 (NEWS2) in the triage section of the ED. Hence, scoring of NEWS2 is part 
of clinical routine at Akershus University Hospital and therefore not a study defined variable. For the ACE 4 Study, we 
will define tachypnea as respiratory rate ≥20/ min according to the NEWS2 scoring on admission in the ED. We apply 
for permission to establish a real-time electronic surveillance of all patients hospitalized at the Division of Medicine, 
Akershus University Hospital that have tachypnea as defined by NEWS2 scoring, which is part of clinical routine. Only 
dedicated study personnel will have access to this information, which will be presented to the study team as a list that is 
stored on secure servers. We also apply for permission to check in the EHR of patients with tachypnea whether the 
patients fulfill inclusion criteria and do not meet exclusion criteria of the ACE 4 Study. After the study team has 
identified eligible patients, the study team will obtain written, informed consent from the patient. Subsequently to 
consent, trained study personnel will collect additional information on current and prior medical history by the strategy 
previously successfully used in the ACE 2 Study. This information will be checked against the EHR of the patient. 
Participants must comply with all inclusion and exclusion criteria to be eligible for study participation.     
All patients will be thoroughly informed about all aspects of the study and the study team will collect informed, written 
consent from all study participants. As a small subgroup of patients with tachypnea will have severely compromised 
clinical status on admission, including possibly hypercapnia that could influence cognitive status, the written informed 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
consent can also be obtained within the first days of the hospitalization after clinical stabilization. In case of severely 
compromised clinical status on admission, the patient will receive oral information on the study and we will limit testing 
to examinations that are indicated from a clinical perspective; e.g. ECG and blood sampling for standard cardiovascular 
(CV) biomarkers. An impartial witness should be present during the entire informed consent discussion, and this will be 
recorded in the patient study file. Informed written consent will be obtained in all subjects as soon as possible after 
stabilization of clinical status. Regardless of the time, at the request by the patient and not already included in a 
publication, all data collected in the study relating to the participant will be terminated. A short run-in phase in the ED 
will be permitted to assess whether the patient will meet the inclusion and exclusion criteria. Blood samples for biobank 
will require signed written informed consent. We will be establishing an eBiobank for our study, which is an electronic 
tracking system for research biobanks approved by the Regional Ethics Committee. It will allow us to keep track of the 
included participants and their respective blood samples in our research biobank. Registration of samples is 
standardized, we will get an overview where the samples are stored, with an overview of sample amount in the 
specimens. Sample search and withdrawals are logged. Sensitive information is encrypted, and the user is working with 
anonymized participant numbers. Each participant can have several samples linked to the eBiobank with unique 
sample numbers. 
The study participants will receive all examinations as considered appropriate by the treating physician, which includes 
also early echocardiography in the control group. There will be no restrictions to the diagnostic testing of our study 
participants, regardless of randomization status in the study. Study personnel will not directly administrate management 
nor therapy of the patients, but the study team will provide structured biomarker-based risk of HF and general 
information related to guideline-based treatment of HF in the study intervention group. The decision of the patient to 
participate in the ACE 4 Study will not influence treatment that is offered to the patient at Akershus University Hospital 
and patients not participating in the study will receive current strategy/ standard care. 
From an ethical perspective the delayed written informed consent can be justified by the argument that most severely 
compromised patients also would benefit most from early detection and treatment of myocardial dysfunction. Hence, 
although we clearly acknowledge the need for all research to adhere to Good Clinical Practice and consider written 
informed consent as foundation for all research, it is imperative that the most severely ill patients can be included into 
the ACE 4 Study. Of note, the non-invasive biomarker measurement is part of routine examinations in this patient group 
and do not in any way put the patient at risk. Accordingly, we consider a delayed written consent in a subgroup of 
patients to be acceptable. The study participants will also receive all additional examinations as considered appropriate 
by the treating physician and study personnel will not dictate diagnostic work up or the plans for the hospitalization or 
follow-up period. Delayed written consent will also only be performed in a small subgroup of patients as we plan to 
obtain written informed consent prior to study inclusion in the majority of our participants. The question regarding 
delayed informed written consent were addressed also by the independent Ethics Committee during formal approval of 
the study and we received support to pursue this strategy. 
3.2 Inclusion Criteria 
 
All the following conditions must apply to the prospective patient at screening prior to inclusion in the study:   
• 
Patients ≥18 years old 
• 
Tachypnea defined as respiratory rate ≥20/ min, as noted in the NEWS 2 classification performed on 
admission in the ED and reported in the patient' EHR, which is performed as routine in all admission at 
Akershus University Hospital (hence, part of clinical routine)   
• 
Admission to Departments under the Division of Medicine at Akershus University Hospital, except the 
Department of Neurology 
• 
<24 h from hospital admittance to inclusion in the study  
• 
Signed written informed consent during the initial phase of the hospitalization 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
3.3 Exclusion Criteria  
Patients with tachypnea will be excluded from the study if they meet any of the following criteria: 
• 
Previously included into the study (in case of patients presenting with a second hospitalization during the study 
period) 
• 
Known or suspected cancer outside of local control, documented in medical records, at the time of patient 
inclusion or diagnosed in relation to the index hospitalization   
• 
Neurological condition with short life expectancy; e.g. ALS, documented in medical records during screening 
prior to study entry 
• 
Other non-cardiac disease with life expectancy below 1 year, documented in medical records during screening 
prior to study entry   
• 
Obvious non-cardiac cause for tachypnea based on medical records and clinical findings during screening 
prior to study entry; e.g. anaphylaxis in young patient with known allergy, dyspnea after direct chest trauma, or 
young patient with fever and positive Covid-19 test on admission. 
• 
Patient assessed as non-Internal Medicine patient; e.g. surgical patient 
• 
Patients unwilling or unable to comply with the protocol, including Glasgow Coma Scale <13 on the time of 
study inclusion 
• 
Patients that are intubated for invasive ventilatory therapy before or shortly after hospital admission 
• 
History of non-compliance to medical management and patients who are considered potentially unreliable, 
based on documentation in medical records, during screening prior to study entry 
• 
History or evidence of alcohol or drug abuse with the last 12 months, based on medical records and clinical 
findings during screening prior to study entry, that will influence study participation  
• 
Any surgical or medical condition, based on medical records and clinical findings during screening prior to 
study entry, that will impair the ability of the patient to participate in the study 
 
3.4 Patient registration  
Each patient in the study will be uniquely identified by a code starting with “ACE4-“ followed in numerical order from 1 
up to the last patient that is included in the study. Once assigned to a patient, a patient number will not be re-used. If 
the patient fails to start on study for any reason, including patients that are randomized and later refuse study 
participation, the reason for not entering the study will be entered in the Screening Log.  
 
  
To ensure that the selected population for the ACE 4 Study is representative to all patients hospitalized with tachypnea 
during the same period, we ask permission to collected basic, anonymized information related to the patients eligible for 
inclusion, but not included into the study (e.g. not enough resources to include all patients on a given day). For these 
patients we will only collect the basic information age, sex, and hospital length of stay for the index hospitalization, and 
we will anonymize the data before storage (i.e. not possible to correlate data to individual patients). 
4 
STUDY EXECUTION 
4.1 Patient Screening, Inclusion, and Randomization 
We will include only patients hospitalized with tachypnea at Akershus University Hospital, location Lørenskog. The 
identification of patients eligible for the study will be directly from automatic, real-time surveillance by the data 
warehouse of Akershus University Hospital of patients with tachypnea, as recorded by ED nurse during assessment of 
NEWS2 scoring and recorded in the patient' EHR (Figure 2). 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
 
Randomization will be performed by a pre-determined computer algorithm that is set up by a statistician. Clinical 
characteristics will be collected directly from the physician and from the medical records by a standardized protocol and 
plotted into the electronic case reports forms (eCRFs) of the study participants.    
4.2 Early Biomarker-based Cardiological Assessment  
The early cardiological assessment will be performed in the subgroup of patients randomized to this intervention 
(Figure 2). Using admission concentrations of NT-proBNP and hs-cTnT, we will use standardized criteria to assess risk 
of HF as the cause for hospitalization. Based on data from the ACE 2 Study and current recommendations for 
diagnosing acute HF, we will use the structured algorithm in Figure 3, which will only be distributed to the study team. 
The values for NT-proBNP are based on contemporary cutoffs for diagnosing HF in the ED setting, with NT-proBNP 
<300 ng/L largely excluding HF, and values above age-specific cutoffs increasing the likelihood of HF being the cause 
of respiratory distress.1 For hs-cTnT, the cutoffs are based on the positive and negative predictive values from ACE 2 
Study, with negative predictive values for HF >90% for hs-cTnT <10ng/L and positive predictive values for HF >90% for 
hs-cTnT ≥90ng/L for patients with intermediate concentrations of NT-proBNP. 
In patients assessed to be in the "grey zone" after as determined by concentrations of NT-proBNP and hs-cTnT, where 
additional examinations are needed to assess risk of HF, and in the patients deemed to be high-risk patients for HF 
based on biomarker testing ("rule in"-group), we will recommend that the patient should perform TTE, if not recently 
performed. According to established guidelines, we will also recommend that an elevated cardiac troponin 
measurement should be followed by additional troponin testing (serial testing), which is required to make the diagnosis 
of acute myocardial infarction.  
 
 
Figure 2. Flow chart of the ACE 4 Study. HF, heart failure. TTE, transthoracic echocardiogram. 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
 
Figure 3. Early cardiological assessment in patients with acute tachypnea. Cutoffs for NT-proBNP are derived from Januzzi JL, et 
al, Journal of the American College of Cardiology 2018;71:1191-200, cutoffs for hs-cTnT in the grey zone are based on results from 
the ACE 2 Study, and the cutoff hs-cTnT ≥14 ng/L is the 99-percentile in the healthy population.  
4.3 Patient Follow-Up During the Index Hospitalization and Data Storage 
All patients that might prove eligible for inclusion in the ACE 4 Study will be identified through automatic, real-time 
surveillance from the data warehouse of Akershus University Hospital. This screening will provide a list of all subjects 
scored with respiratory rate ≥20/min, as noted in the NEWS2 classification performed on admission in the ED and 
reported in the patient EMR. We have identified that median 13 patients/ day (range 2-30 patients) fulfill this first 
inclusion criteria at Akershus University Hospital, location Lørenskog. Only dedicated study personnel will have access 
to this screening log and will then, based on EHR notes, make a first assessment whether the patient might be eligible 
for inclusion into the study. Study personnel will subsequently contact the patient to see whether the patient is 
compliant with the inclusion and exclusion criteria of the ACE 4 Study, provide information regarding the ACE 4 Study, 
and ask whether the patient will provide written informed consent. Study personnel will also consider whether delayed 
consent is more appropriate in patients who are considered to be in a severe clinical situation. Participants will be 
randomized with an electronic algorithm using block randomization and the randomization scheme will not be revealed 
to study personnel. Preferably, only patients with signed written consent will be randomized. Patients that are 
randomized but do not provide consent or later are deemed non-compliant with inclusion or exclusion criteria, will be 
marked as screening failures and not included into the ACE 4 Study.      
We will perform cardiac biomarker testing with NT-proBNP (ProBNP assay, Roche Diagnostics, Basel, Switzerland) 
and hs-cTnT (Elecsys TnT hs stat, Roche Diagnostics) measurements on ED admission in all participants, regardless 
of randomization status. The results will be provided in the patient's EHR, regardless of randomization status in the 
ACE 4 Study. We will provide a note in all patient's EHR that the patient is participating in the ACE 4 Study, has signed 
a written informed consent, and we will provide an overview of the study protocol. In addition, and only for patients 
randomized to the intervention group, we will include assessment of probability that myocardial injury or dysfunction 
are the underlying pathophysiology responsible for tachypnea in the patient' EHR, as evaluated by the cardiac 
biomarker algorithm of the ACE 4 Study (Fig. 3). Furthermore, in patients randomized to the intervention group, we 
will also inform on general recommendations for work up and treatment, but no specific instructions for individual 
patients as the treating physician should make all clinical decisions.  
The study participants will receive all examinations considered appropriate by the treating physician, including TTE in 
the control group if this is considered appropriate from a clinical perspective. Hence, there will be no restrictions to 
diagnostic testing of our study participants, regardless of randomization status. If requested by the treating physician, a 
full TTE will be performed by trained study personnel in both groups. The TTE protocol will assess atrial and ventricular 
dimensions, systolic and diastolic properties of the left ventricle, right ventricular systolic function, the cardiac valves, 
pericardial effusion, and estimate arterial pulmonary pressure + look for other signs indicative of pulmonary 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
hypertension. The conclusion of the TTE will be recorded in the patient' EMR, according to standard procedure. The 
decision of the patient to participate or to decline participation into the study will not influence the treatment that is 
offered to the patient at Akershus University Hospital and also patients declining participation will receive current 
strategy/ standard care. 
 
After written, informed consent from the patient, trained study personnel will collect additional information on prior 
medical history by the strategy previously used in the ACE 2 Study. This information will be checked against the EHR of 
the patient. All participants of the study, regardless of randomization status, will be subjected to extensive CV 
phenotyping, including blood sampling for biobank (admission, discharge, and after 6 months), and short-term ECG 
monitoring (≤24 h). Furthermore, and only in patients not receiving TTE early after admission as part of clinical work-
up, we will perform a TTE for research purposes prior to hospital discharge. Hence, all participants of the ACE 4 Study 
will be examined by TTE during the index hospitalization. The results of TTE performed for research purpose late 
during the hospitalization will not be communicated to the treating physician, unless we identify unrecognized, gross 
pathology like left ventricular ejection fraction<40%, right ventricular strain indicative of possible pulmonary embolus, 
severe valvular disease, or pericardial effusion with risk of cardiac tamponade. 
 
We will not provide any treatment recommendation for individual patients. However, we will include a short paragraph 
related to information for pharmacological treatment of patients with HFrEF (LVEF≤50%), HFmrEF (LVEF 40-50%), 
and HF with recovered LVEF according to the ESC Heart Failure Guidelines 2021. For these patients we will inform the 
physician that β-blocker, SGLT2-inhibitor, inhibition of renin-angiotensin with ACE inhibitors or angiotensin-neprilysin 
inhibition, and aldosterone inhibition is recommended. We will also inform that loop diuretics are recommended for 
volume control/ decongestion in all patients with HF according to the ESC guidelines. The recent ESC guidelines have 
no clear pharmacological recommendations for patients with LVEF≥50% (HFpEF), but based on emerging data from 
randomized clinical trials demonstrating effects from SGLT2-inhibitors across the spectrum of LVEF in HF patients (e.g. 
DELIVER trial) we will inform the treating physician about SGLT-2-inhibitiors in HFpEF patients. We will also inform the 
treating physician about aldosterone inhibition, as aldosterone inhibition reduced HF hospitalizations in the TOPCAT 
trial (secondary endpoint) and reduced the primary endpoint of TOPCAT in post-hoc analysis. The recent US 
(AHA/ACC/HFSA) guidelines for heart failure (2022) supports this strategy. 
 
We will also inform that, according to several ESC guidelines, patients with suspected acute coronary syndromes are 
recommended to be clinically assessed for coronary artery disease and treated according to current guidelines. We will 
also, on a general basis, inform the treating physician that patients with atrial fibrillation have guideline-
recommendations for treatment, including anti-coagulation and frequency control. 
  
Patients will be followed up during the index hospitalization for all endpoints related to the hospitalization. We will 
record the time in hospital, and the time spent in high-resource units (Intensive Care Unit/Medical Intensive Care 
Unit/Cardiac Intensive Care Unit). We will also record all additional diagnostic testing performed in the patients and 
calculate the total resource utilization for the individual patient. We will also collect data on 30-day all-cause 
readmission from the data warehouse of Akershus University Hospital and from national registries. We will use similar 
strategy to collect data also for secondary and exploratory endpoints.  
4.4 Adjudication of Diagnosis and Clinical Events 
The diagnosis of the index hospitalization will be adjudicated by two experts working independently and reviewing all 
data, including follow-up data, as previously also performed in the ACE 2 Study and other international studies. For the 
ACE 4 Study the experts will have access to the results of CV biomarkers measured during the hospitalization and to 
the results of TTEs, however, the experts will not have direct access to the conclusion of the early cardiological 
assessment. A diagnosis of HF as the cause for the hospitalization will be based on the criteria proposed by ESC 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
requiring typical signs and symptoms of HF and objective evidence of structural or functional myocardial abnormality, 
most recently updated with ESC Guidelines for Heart Failure from 2021. Discrepancy regarding the diagnosis between 
the two experts will be resolved by consensus and we will document the proportion of cases where the experts initially 
do not agree.  
For follow-up we will collect all data and any hospitalization, regardless of illness and diagnosis (all-cause morbidity and 
all-cause mortality). A diagnosis of acute myocardial infarction will be based on the definition by the most recent WHO 
Universal Definition for myocardial infarction; i.e. (1) Either patient history suggestive of acute myocardial infarction 
(AMI) but sudden death before blood samples could be obtained OR (2) troponin rise and/or fall. In addition, one of the 
following criteria also needs to be present: (i) Symptoms suggestive of acute coronary syndrome (ACS), (ii) ECG signs 
of AMI, (iii) TTE signs of AMI, or (iiii) obstructive coronary artery disease on angiogram. Exacerbation of chronic 
obstructive pulmonary disease (AECOPD) will be defined by the Global initiative of chronic Obstructive Lung Disease 
(GOLD) criteria: “[…] a worsening of the patient’s symptoms [dyspnea, cough and/or sputum production] that is beyond 
day-to-day variation and that leads to a change in medication.” COPD will require pulmonary testing with FEV1/FVC 
<0.7 and we will use the GOLD category 1-4 according to FEV1. Pulmonary embolus (PE) will be diagnosed based on 
evidence of PE on CT scan (or other modality), or TTE evidence of TTE in the absence of other known cause for right 
ventricular strain/other indices possibly reflecting PE. Pulmonary infections will be diagnosed based on clinical 
symptoms of infections (e.g. sputum production, fever, etc), biomarkers reflective of inflammation during the 
hospitalization and/or chest X-ray/CT scan demonstrating pulmonary infection. Mortality during follow-up will be 
collected from the data warehouse of Akershus University Hospital and national registries.     
4.5 Quality of Life and Resource Utilization 
We will use the EQ-5D-5L questionnaire as basis for calculating health related quality of life (HRQoL) in our study 
participants. Information from EQ-5D-5L will be collected soon after admission and prior to discharge during the index 
hospitalization. Planned time points for QoL assessment are at discharge, after 1 month, 3 months, 6 months, 12 
months, and yearly afterward for maximum 10 years. 
Financial resource utilization will be performed by collecting information on hospital length of stay and type of hospital 
ward (ICU/CCU vs. standard) and by collecting information on all supplementary tests performed during or in 
conjunction with the index hospitalization. To calculate total costs during follow-up we will obtain information on hospital 
re-admissions during follow-up, medication use, sick leave from work, and nursing home care. We will obtain this 
information from hospital systems (re-admissions) and regional and national registries (medications [national 
medication registry], sick leave [NAV/SSB], and nursing home use [IPLOS]). We will also seek to obtain data on 
medical contacts with primary care physicians from national registries [KUHR/KPR] and travel costs for patients. In 
addition, we will also obtain information from other registries (e.g. FD-trygd and Norwegian Patient Registry) to inform 
resource use in the health economic evaluation. 
4.6 Deep Phenotyping and Exploratory Endpoints 
There is a need to understand more closely the pathophysiology and comorbidities of the major conditions causing 
hospitalization for acute dyspnea. Accordingly, in the ACE 4 Study we will also characterize all participants with TTE, 
ECG monitoring in the acute phase, and collection of biospecimens that later can permit quantification of protein and 
non-protein biomarkers. We will collect blood samples for study biobank from all participants within 24 h from hospital 
admission and before hospital discharge (Figure 2). We will invite included patients back for a 6-month outpatient visit 
to register medication and to collect biospecimen for biobank storage. The rationale for biospecimen collection also 
after 6 months is to assess serial concentration of biomarker over time, and to correlate changes in biomarker 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
concentration with medication. Blood sampling will be performed by standard venous access or from indwelling arterial 
cannula if already present due to clinical need. We will obtain serum and citrate, heparin, and EDTA plasma.  
We will measure established and novel CV protein and non-protein biomarkers. Categories of biomarkers that will be 
measured relate to markers of hemodynamic stress, myocardial injury and function, neurohormones, cell necrosis, 
fibrosis, inflammation, renal disease, and pulmonary or other non-cardiac organ status. In addition, we ask permission 
to use -omics based methodology to perform unbiased analyses of RNA, protein, and metabolites. The revised protocol 
will be reviewed by the Regional Ethics Committee prior to the start of the ACE 4 Study. We ask permission to send 
samples to collaborating researchers outside of Akershus University Hospital and Norway. Possible countries that may 
receive samples are all Western European countries, but especially Sweden, Denmark, Finland, Germany, Great 
Britain, Switzerland, Italy, and Austria. We also ask permission to send samples outside of Europe, including to 
countries with different legislation compared to Norway for biological samples, and the main recipients are UK, USA, 
Australia, China, and India. If samples are sent out from Akershus University Hospital the collaborating center will only 
receive de-identified samples; i.e. no samples will be sent out with linked personal information so that the material can 
be linked back to individual patients of the ACE 4 Study.  
Clinical phenotyping will be performed by obtaining short-term continuous ECG monitoring (≤24 h) during day 1 after 
patient inclusion into the study in all subjects, regardless of randomization status. This will be performed by the use of 
portable ECG monitors (Holter recordings) and the ECG recordings will be assessed by trained research personnel. 
Possible traits related to the ECG recordings are AF, ventricular ectopies, complex ventricular ectopies, non-sustained 
ventricular tachycardias, and sustained ventricular tachycardias. We will also obtain data on indices of heart rate 
variability. Also data from these recordings may be shared with national and international collaborators analogous to 
the countries listed above for biological analysis. Only de-identified data will be sent out from Akershus University 
Hospital.  
We will perform a full TTE examination in all subjects during the hospitalization, regardless of initial randomization 
status. TTE will be performed by the Vivid E95 (GE VingMed, Horten, Norway) and the images will be digitally stored 
for offline analysis on custom software (EchoPac, GE VingMed). We will acquire 2D images and loops with a 2.5 MHz 
transducer and 3D images with a 4 V matrix-array transducer. Standard parasternal long axis and three apical views 
recordings will be done in the end-expiratory phase with the subjects in supine left lateral position. LV dimensions, 
septal and posterior wall thickness, and LV mass will be measured as recommended by American Society of 
Echocardiography. LV ejection fraction (EF) will be calculated using the modified Simpson’s rule from biplane 4-
chamber and long-axis views. LV diastolic function will be assessed by pulsed Doppler transmitral peak early (E), peak 
late (A) and E deceleration time. TVI-derived indices will be recorded at the base of the septal and lateral mitral annulus 
to determine peak systolic (S’), early diastolic (e’) and late (a’) diastolic velocities. Global and regional longitudinal strain 
will be analyzed by an offline semi-automated speckle tracking technique from the three apical views. The digital 
storage of echocardiographic images in Echopac will permit later analyses of novel imaging indices. The 
echocardiographic recordings are obtained by a limited number of researchers, and we will ensure standardization of all 
recordings. The results of the TTE considered standard in the art and available at the time of the examination, will be 
recorded in the medical records of the patient, but will not be communicated directly to the physician of the patient 
except in cases of gross pathology as outlined in section #4.3. Also data from these recordings may be shared with 
national and international collaborators analogous to the countries listed above for biological analysis. Only de-
identified data will be sent out from Akershus University Hospital.   

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5 
DATA MANAGEMENT AND STATISTICAL ANALYSIS 
5.1 Electronic Case Report Forms (eCRFs) 
Case report forms (CRF) will be provided for the recording of all data and we will use RedCap for data storage. Data 
will be recorded directly and legibly onto the eCRF and verified by the investigator. If any assessments are omitted, the 
reason for such omissions will be noted on the eCRFs. Corrections, with the reason for the corrections if applicable, 
should be dated and initialed. Data will be transferred to a secure area on TSD, University of Oslo. Study participants 
will only be identified by the study ID in the eCRF and the key to connect the eCRF data to the identity of the patients 
will be stored in a secure place with access control (only accessible to the PI and dedicated members of the study 
team). 
5.2 Source Data  
We will automatically monitor all admissions through the ED at Akershus University Hospital meeting the inclusion 
criteria of tachycardia, as documented in the respiratory variable of the NEWS2 score. Of note, NEWS2 scoring is 
performed routinely in all admissions at Akershus University Hospital for clinical purposes and therefore not a study 
specific action for the ACE 4 Study. Only study team members will have access to this screening list and these data will 
only be available on safe data systems connected to the data warehouse at Akershus University Hospital. Members of 
the study team will access the patient' EHR of patients that have been identified by the automatic surveillance to make 
initial assessment regarding study eligibility. We will record data on the number of patients detected by the screening 
log and prevalence of these patients that are approached for possible study inclusion, but no other information will be 
stored related to the initial, automatic surveillance for eligible patients. The study team also apply for permission to 
access the patient' EMR to determine whether the patient may be eligible for participation in the ACE 4 Study.   
For patients that are included in the ACE 4 Study, we will review all patient' EHR and perform a structured interview of 
study participants, which is analogous to the strategy used in the ACE 2 Study. Data obtained during the hospitalization 
(Figure 2) will also be collected, de-identified, and stored in a secure web solution. Only de-identified data (marked with 
study code) will be shared with national and international collaborators as outlined in section #4.5. We also ask 
permission to collect some standard, demographic data on the patients that are found eligible for study inclusion, but 
decline to participate in the ACE 4 Study. We believe some key information on age, gender, and hospital length of stay 
are relevant to report for non-included patients to document that the final study population is representative for the total 
population that could have been included into the study.   
We will ask permission to link our data to registries and the hospital records during follow-up. Morbidity and mortality 
data will be obtained from the data warehouse at Akershus University Hospital and from national registries.   
5.3 Source Data Verification 
The investigator will be visited on a regular basis by a Clinical Study Monitor, who will check and collect completed 
eCRFs, discuss the progress of the study, and perform source data verification. 
When the responsible study monitor has checked and verified the CRFs, the data will be entered into a computer 
database at TSD, University of Oslo for further handling and statistical evaluation. 
Sponsor’s representatives (e.g. monitors, auditors) and/or regulatory authorities will be allowed access to source data 
for source data verification in which case a review of those parts of the hospital records relevant to the study may be 
required. 

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5.4 Storage of Study Documentation 
The investigator shall arrange for the retention of the patient identification and the code list. Patient files shall be kept 
for the maximum period of time permitted by each hospital. The study documentation (eCRFs, Site File etc) shall be 
retained and stored during the study and for 5 years after study closure, which was reported as a requirement from the 
Regional Ethics Committee when granting permission for study start. All information concerning the study will be stored 
in a safe place inaccessible to unauthorized personnel.  
5.5 Study Design  
This is a single center, pragmatic randomized controlled trial of a non-pharmacological intervention. 
The data will be summarized with respect to demographic and baseline characteristics and efficacy observations and 
measurements. Categorical data will be presented as absolute frequencies and percentages. For continuous data, N, 
median, and quartile 1-3 will be presented. Time to event variables and Kaplan-Meier product-limit estimates will be 
presented for the primary endpoint and other endpoints. The primary analyses will be conducted on all patient data at 
the time all patients have been included in the study and after hospital discharge of the last patient (regarding the index 
diagnosis). The following populations will be used for analysis: 
 
• 
Intention to Treat Analysis. Contains all patients of the population that were included in the study and 
randomized to early structured biomarker assessment or standard care.  
• 
Per-protocol population. Consists of all patients of the intent-to-treat population who show no major protocol 
violations, i.e. violations that may have an impact on the study outcome. 
 
5.6 Sample Size & Statistical Considerations 
Statistical power calculations are based on data from the ACE 2 Study, which included 314 patients with acute dyspnea 
using a similar strategy as employed for the ACE 4 Study. The power calculations are based on the assumptions that 
approximately 45% of patients hospitalized with dyspnea will be diagnosed with acute HF and that a substantial 
proportion of the patients with non-HF related dyspnea will also exhibit evidence of myocardial dysfunction; in total 65% 
of the ACE 2 cohort was classified as hospitalized with myocardial dysfunction. The 1-year readmission/all-cause 
mortality rate in the ACE 2 Study was approximately 40%, and we expect a rate reduction to 25% in the early 
cardiological intervention group among the patients with myocardial dysfunction. Based on these numbers and using a 
two-sided test, we will need to include 152 patients in the control group and 152 patients in the early cardiological 
assessment group to have 80% probability to detect a significant difference (p<0.05). Based on data from the ACE 2 
Study, 65% of the population will suffer from myocardial dysfunction, thus we will need to randomize 234 patients to 
early cardiological assessment. With a 1:1 randomization strategy taking into account possible patient dropouts, we will 
aim to include at least 500 patients in the ACE 4 Study. However, we ask permission to include up to 600 patients if 
additional pilot experiments prior to study commencement indicate a need for a larger sample size. 
The study will be analyzed on an intention-to-treat basis. That includes all patients enrolled in the study, irrespective if 
they have received the intervention or not. As a scenario analysis, we will also assess all outcomes by per-protocol 
analysis (i.e. including only the patients that actually received the intervention into the analysis).  
5.7 Safety Analysis 
The assessment of safety will be based mainly on the frequency of Adverse Events during the index hospitalization. 
Adverse Events will be summarized by presenting the number and percentage of patients having any adverse event by 

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body system and type of Adverse Event. Those Adverse Events that result in death or life-threatening event will be 
presented separately. We will include a Data Safety and Monitoring Board (DSMB) that will review the data for safety 
signals after inclusion of 100, 200, and 400 patients. The DSMB will assess key data like primary and some secondary 
endpoints in the two groups. The DSMB may then recommend that the study continues or stops based on safety or 
futility.  
5.8 Interim Analysis 
We will not perform interim analysis. 
 
6 ETHICAL AND REGULATORY REQUIREMENTS 
6.1 Ethical Considerations 
6.1.1 General Considerations 
The responsible investigator will ensure that this study is conducted in agreement with the Declaration of Helsinki 
(Tokyo, Venice, Hong Kong, Somerset West and Edinburgh amendments) and the laws and regulations of the country 
where the trial is performed. The protocol has been written, and the study will be conducted according to the ICH 
Harmonized Tripartite Guideline for Good Clinical Practice (ref: http://www.ifpma.org/ethics/ifpma-code-of-
practice/about-ifpma-code-of-practice.html). The study will be evaluated by the Regional Ethics Committee and other 
government agencies before initiation. The protocol will be registered in www.clinicaltrials.gov before inclusion of the 
first patient. 
6.1.2 Informed Consent 
All patients will be thoroughly informed about all aspects of the study, including the examinations during the index 
hospitalization, and the required evaluations for informed consent, which will be obtained during or just after hospital 
admission. The patients will also be informed that we will obtain information from national health registries and also 
information from other health care provides. As a small subgroup of patients with acute tachypnea have severely 
compromised clinical status on admission, including hypercapnia that may reduce cognitive status, the written informed 
consent can also be obtained within the first days of the hospitalization after stabilization. In case of severely 
compromised clinical status on admission, the patient will receive oral information on the study and we will limited 
testing to examinations that may also be indicated from a clinical perspective; e.g. ECG and blood sampling for 
standard CV biomarkers. An impartial witness should be present during the entire informed consent discussion and this 
will be recorded in the patient study file. Informed written consent will be obtained in all subjects as soon as possible 
after stabilization of clinical status. Regardless of the time, at the request by the patient and if data are not already part 
of a publication, all data collected in the study relating to the particular participant will be terminated. A short run in 
phase in the ED will be permitted to assess whether the patient meet the inclusion and exclusion criteria. Blood 
samples for biobank will require signed written informed consent. From an ethical perspective the delayed written 
informed consent can be justified by the argument that the most severely compromised patients also, potentially, will 
have the largest benefit of early detection of myocardial dysfunction. Hence, although we clearly acknowledge the need 
for all research to adhere to Good Clinical Practice and consider written informed consent as a foundation for all 
research, the non-invasive strategies implemented in our early structured biomarker assessment is part of routine 
examinations in this patient group and in no way put the patient at risk. Accordingly, we consider a delayed written 
consent in a subgroup of patients to be acceptable. Of note, we will obtain written informed consent in the large majority 
of patients prior to patient inclusion and randomization, thus, delayed written consent will only be performed in a small 
subgroup of patients. However, as this subgroup also will be the most severely ill patients, including them also in the 

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study will be important for internal and external validity of the study, and as this subgroup potentially may benefit the 
most from early structured biomarker assessment. 
It will be emphasized that the participation is voluntary and that the patient is allowed to refuse further participation in 
the protocol whenever he wants. This will not prejudice the patient’s subsequent care and all patients, regardless of 
study participation, will receive current strategy/ standard care. Documented informed consent must be obtained for all 
patients included in the study before they are registered for the study. This must be done in accordance with the 
national and local regulatory requirements. 
The written informed consent form should be signed and personally dated by the patient or by the patient’s legally 
acceptable representative. 
To ensure that the selected population for the ACE 4 Study is representative to all patients hospitalized with tachypnea 
during the same period, we ask permission to collected basic, anonymized information related to the patients eligible for 
inclusion, but not included into the study (e.g. not enough resources to include all patients on a given day). For these 
patients we will only collect the basic information age, sex, and hospital length of stay for the index hospitalization, and 
we will anonymize the data before storage (i.e. not possible to correlate data to individual patients). 
6.2 Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in accordance with the Declaration 
of Helsinki and Good Clinical Practice (ICH-GCP). The Principal Investigator is responsible for personally overseeing 
the treatment of all study patients. The Principal Investigator must assure that all study site personnel, including sub-
investigators and other study staff members, adhere to the study protocol and all local and federal regulations and 
guidelines regarding clinical trials both during and after study completion. 
The Principal Investigator is also responsible for assuring that all the required data will be collected and entered onto 
the CRFs. Periodic monitoring visits will be conducted and the Principal Investigator will provide access to his/her 
original records to permit verification of proper entry of data. At the completion of the study, all case report forms will be 
reviewed by the Principal Investigator and will require his/her final signature to verify the accuracy of the data. 
6.3 Record Retention 
Study documentation includes all CRFs, data correction forms or queries, source documents, Sponsor-Investigator 
correspondence, monitoring logs/letters, and regulatory documents (e.g., protocol and amendments, IRB 
correspondence and approval, signed patient consent forms). Source documents include all recordings of observations 
or notations of clinical activities and all reports and records necessary for the evaluation and reconstruction of the 
clinical research study. 
Government agency regulations and directives require that the study investigator must retain all study documentation 
pertaining to the conduct of a clinical trial. Study documents should be kept on file for 15 years after the completion and 
final study report. 
6.4 Audits 
To ensure the quality of data, study integrity, and compliance with the protocol and the various applicable regulations 
and guidelines, the sponsor may conduct site visits to institutions participating in this study. The investigator, by 
accepting to participate to this protocol, agrees to co-operate fully with any quality assurance visit undertaken by third 
parties, including representatives from the sponsor as well as to allow direct access to documentation pertaining to the 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
clinical trial (including CRFs, source documents and other study files) to these authorized individuals. The investigator 
must inform the sponsor immediately in case of a scheduled inspection by a regulatory authority. 
6.5 Publication Policy 
The findings of this study will be published independent of its outcome. All personnel who have contributed significantly 
with the planning or to perform the study (Vancouver convention 1988) may be included in the list of authors. 
7 
STUDY MANAGEMENT 
7.1 Investigator Delegation Procedure 
The Principal Investigator is responsible for making and updating a “delegation of tasks” listing all the involved co-
workers and their role in the project. He will ensure that appropriate training relevant to the study is given to all of these 
staff, and that any new information of relevance to the performance of this study is forwarded to the staff involved. 
7.2 Study Amendments 
If it is necessary for the study protocol to be amended, the amendment and/or a new version of the study protocol 
(Amended Protocol) must be notified to and approved by the Competent Authority and the Ethics Committee according 
to EU and national regulations. 
7.3 Audit and Inspections 
Authorized representatives of a regulatory authority and Ethics Committee may visit the center to perform inspections, 
including source data verification. Likewise, the representatives from the sponsor may visit the center to perform an 
audit. The purpose of an audit or inspection is to systematically and independently examine all study-related activities 
and documents to determine whether these activities were conducted, and data were recorded, analyzed, and 
accurately reported according to the protocol, Good Clinical Practice (ICH GCP), and any applicable regulatory 
requirements. The Principal Investigator will ensure that the inspectors and auditors will be provided with access to 
source data/documents. 
8 
TRIAL SPONSORSHIP AND FINANCING 
The study is sponsored by Akershus University Hospital with financial support from Helse Sør-Øst to a PhD position. 
9 
TRIAL INSURANCE 
This study is covered by the general insurance of Akershus University Hospital.   
10 
ACKNOWLEDGEMENTS 
A study protocol for a clinical trial of patients hospitalized with acute dyspnea as the inclusion criterion was initially 
developed in 2014-2015 by Prof Helge Røsjø in collaboration with Drs. Aagaard, Kvisvik, Caspersen, and Gravning. 

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ACE 4 Study Protocol Version No.3, December 3, 2023 
The initial study protocol was never used for patient inclusion. Hence, Prof Røsjø in collaboration with Ass Prof 
Lyngbakken and R. Bhatnagar MD, Kristian Berge MD, and Professor Henrik Schirmer performed a revision of the 
protocol in 2021 and 2022 where inclusion criteria, screening strategy, intervention, and endpoints were revised. 
Hence, we consider version #2 to represent a revised de novo protocol and we will apply for renewed approvals from 
the Regional Ethics Committee and Data Protection Officer at Akershus University Hospital. Torbjørn Wisløff ,PhD, also 
contributed to the de novo revised protocol. The total study group, which is reported on the front page of the protocol, 
have also contributed to the new protocol by providing oral and written suggestions to the revised protocol (version #2).      

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ACE 4 Study Protocol Version No.3, December 3, 2023 
11 
APPENDIX 
11.1 Echocardiographic protocol  
ACE 4 – Akershus Cardiac Examination 4 Study 
 
ECHOCARDIOGRAPHIC RECORDING PROTOCOL: 
Required views for 2nd echo (approx. 30 min) 
 
The number of loops and images to be recorded is minimum numbers (fifth column). All loops and images will be 
recorded in 50 mm/sec with the participants in supine and left lateral decubitus position at the end of expiration. The 
echo machine has to be set to “Cardiac mode”, which is important for the 2D strain recordings. Also important for 
2D strain analysis is to include all LV and LA endocardium (must be visible). Blood pressure in supine position 
between echo and linear recordings. 
 
No 
Projection 
Mode 
Measurements to perform. Comments 
Recordings 
1 
PLAX 
2D 
Left atrium (LA), Left ventricle (LV) and LV 
outflow tract 
4 loops 
2 
PLAX 
M-mode 
LV dimensions, LV mass 
1 image with 
at least 5 HS 
3 
PSAX 
2D 
Presentation of the aortic valve, three or bi-leaflet 
4 loops 
4 
PSAX 
2D 
At the papillary muscle level 
4 loops 
(4) 
PSAX 
M-mode 
LV dimensions, LV mass. Recording only 
necessary if PLAX, M-mode (#2) not good 
1 image with 
at least 5 HS 
5 
Apical 4- 
chamber 
(4CH) 
2D 
LV, RV, LA and RA. Measure: end systolic (es) 
and end diastolic (ed) volumes of LV (4C and 
2C ad mode Simpson`s), es volume of VA (areal 
- length, biplane, 4C and 2C). 
2-D strain systolic measurements of LV, RV and LA. 
(Important: endocardium must be visible and framerate per 
second between 40 – 90) 
4 loops 
(Often 4 loops 
x 2, if 
RV and RA 
are not good 
enough 
recorded) 
6 
4CH 
Pulsed 
Doppler (PW) 
Transmitral early (E) and late (A) for the 
assessment of LV diastolic function. Peak velocity of E 
and A and deceleration time of E. 
2  images 
with at least 
5 HS 
7 
4CH 
M-mode 
TAPSE (tricuspid annular plane systolic 
excursion), which reflect RV systolic function. 
1 image with 
at least 5 HS 
8 
4CH 
Cont. 
wave
Doppler (CW) 
Pressure gradient between RV and RA 
1 image with 
at least 5 HS 

36 
 
 
9 
4CH 
Tissue 
velocity 
imaging 
(TVI*) 
Possibility to measure Systolic and diastolic TVI 
velocities in LA, LV, RA and RV: Frame rate 
>100/s.  See point 4 regarding RA and RV 
4 loops 
(often 
4 
loops x 2 
10- 
11 
4CH 
TVI* +  PW 
Systolic S, diastolic E` an A` at septal and 
lateral mitral attachment. 
2  images 
with at least 
5 HS 
12- 
13 
4CH 
TVI* +  PW 
Systolic S, diastolic E` and A` at lateral part of 
tricuspid valve and isovolumetric relaxation time. 
2  images 
with at least 
5 HS 
15 
Apical 2- 
Chamber 
(2CH) 
2D 
Volumes of VV and VA for the 2D strain 
analysis. 4 loops x 2 if VA not good recorded 
4 loops 
16 
2CH 
TVI* 
TVI with frame rate >100/s 
4 loops 
17 
Apical 
long-axis 
(APLAX) 
2D 
Volumes of VV and VA for the 2D strain 
analysis. 
4 loops 
18 
APLAX 
TVI* 
TVI with frame rate >100/s 
4 loops 
19 
4CH 
3D 
Volumes of VV and VA for the 3D strain. 
4 loops extra if VA not good recorded 
4 loops 
20 
2CH 
3D 
Volumes of VV and VA for the 3D strain. 
4 loops extra if VA not good recorded. 
4 loops 
21 
APLAX 
3D 
Volumes of VV and VA for the 3D strain. 
4 loops extra if VA not good recorded 
4 loops 
 
*All TVI projections may be divided into 2 or more recordings if needed for frame rate >100/s. 
 
See separate description of additional echo recordings by pathological findings. 
 
12 
REFERENCES 
1. Januzzi JL, et al. N-terminal pro–b-type natriuretic peptide in the emergency department. Journal of the American 
College of Cardiology 2018;71:1191-200.