The Conservative vs. Liberal Approach to Fluid Therapy of Septic Shock in Intensive Care Trial
Phase: PHASE4
Conditions: Septic Shock
Sponsor: Anders Perner, MD, PhD

 
 
 
 
 
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Effects of restricting intravenous fluids vs. standard care fluid 
therapy in patients with septic shock 
 
 
The Conservative vs. Liberal Approach to fluid therapy of Septic 
Shock in Intensive Care (CLASSIC) Trial 
 
 
 
 
 
 
 
 
 
 
Management Committee 
Anders Perner, sponsor 
Tine S Meyhoff, coordinating investigator 
Maj-Brit Nørregaard Kjær, trial manager 
Peter B Hjortrup, initiator 
Morten Hylander Møller, clinical trialist 
Theis Lange, statistician 
Jørn Wetterslev, trialist 
 
Protocol version and date 
2.4, 23/06/2022 
 
Applicable protocol registration numbers 
ClinicalTrials.gov identifier NCT03668236 
Ethics committee number H-18006255 
EudraCT number 2018-000404-42 
Danish Medicines Agency number 2018020596 
 
 
 
The Copenhagen Trial Unit’s Standard Operating Procedures were used for this protocol 

 
 
 
 
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Abstract 
• Background. Septic shock is common, often lethal, costly, and associated with prolonged suffering 
among survivors and relatives. Traditionally, intravenous (IV) fluids are used to optimise the 
circulation, and the use of higher volumes is recommended by international guidelines. There is, 
however, no high-quality evidence to support this. In contrast, data from cohort studies, small 
trials and systematic reviews in sepsis and large trials in other settings and patient groups suggest 
potential benefits from restriction of IV fluids in patients with septic shock. 
• Objectives. We aim to assess the benefits and harms of IV fluid restriction vs. standard care on 
patient-important outcome measures in adult intensive care unit (ICU) patients with septic shock. 
• Design. CLASSIC is an international, multicentre, parallel-grouped, open-labelled, centrally 
randomised, stratified, outcome assessor- and analyst-blinded trial. 
• Inclusion and exclusion criteria. We will screen all adult ICU patients who have septic shock 
defined according to the Sepsis-3 criteria and have received at least 1 L of IV fluid (crystalloids, 
colloids or blood products) in the 24-hours before screening. We will exclude patients who have 
had septic shock for more than 12 hours at the time of screening, who have life-threatening 
bleeding, or acute burn injury >10% of the body surface area, who are pregnant and those in 
whom consent cannot be obtained as per the model approved for the specific site. 
• Experimental intervention. In the IV fluid restriction group no IV fluids should be given in the ICU 
unless extenuating circumstances occur, including signs of severe hypoperfusion, overt fluid loss 
or a failing GI tract with a total fluid input of less than 1 L per day. In these circumstances, IV fluid 
may be given in measured amounts. 
• Control intervention. In the standard care group there will be no upper limit for the use of IV 
fluids. 
• Outcomes. The primary outcome is 90-day mortality; secondary outcomes are serious adverse 
events in the ICU (ischemic events or severe acute kidney injury); days alive without life support 
at day 90; days alive and out of hospital at day 90 and mortality, health-related quality of life and 
cognitive function at 1-year. 
• Trial size. We will randomise 1554 participants to allow the detection of a 15% relative risk 
reduction (7% absolute) in the restrictive vs. standard care group in 90-day mortality with a power 
of 80%. 
• Timeline. 
- Primo 2018 Authority approvals in DK 
- Medio 2018 1st participant randomised in DK and authority approvals elsewhere 
- Primo 2019 1st interim analysis 
- Medio 2019 2nd interim analysis 
- Ultimo 2019 3rd interim analysis 
- Medio 2020 Last participant randomised 
- Ultimo 2020 Primary report on 90-day outcomes submitted. 
- Medio 2021 Last participant followed for 1 year 
- Ultimo 2021 Long-term outcome report submitted 
 
 

 
 
 
 
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Administrative information 
Sponsor  
Anders Perner, senior staff specialist and professor in intensive care medicine 
Dept. of Intensive Care 
Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen Ø 
+45 3545 8333 
anders.perner@regionh.dk 
 
Clinical trial sites  
In Denmark 
 
Dept. of Intensive Care 
Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen Ø 
 
Dept. of Anaesthesia and Intensive Care 
Bispebjerg Hospital, University of Copenhagen 
2200 Copenhagen N 
 
Dept. of Anaesthesia and Intensive Care 
Herlev Hospital, University of Copenhagen 
2730 Herlev 
 
Dept. of Anaesthesia and Intensive Care 
North Zealand Hospital, University of Copenhagen 
3400 Hillerød 
 
Intensive Care Unit Q-022GE  
Gentofte Hospital 
2900 Hellerup 
 
Dept. of Anaesthesia and Intensive Care 
Zealand University Hospital, Køge 
4600 Køge 
 
Dept. of Anaesthesia and Intensive Care 
Zealand University Hospital, Roskilde 
4000 Roskilde 
 
Dept. of Anaesthesia and Intensive Care 
Slagelse Hospital 
4200 Slagelse 
 
Dept. of Anaesthesia and Intensive Care 
Randers Hospital 
8930 Randers 

 
 
 
 
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Dept. of Anaesthesia and Intensive Care 
Herning Hospital 
7400 Herning 
 
Dept. of Anaesthesia and Intensive Care 
Viborg Hospital 
8800 Viborg 
 
Dept. of Anaesthesia and Intensive Care 
Aalborg University Hospital 
9000 Aalborg 
 
Dept. of Anaesthesia and Intensive Care 
Lillebælt Hospital 
6000 Kolding 
 
In Finland 
Division of Intensive Care Medicine, 
Department of Anaesthesiology, Intensive Care and Pain Medicine, 
University of Helsinki and Helsinki University Hospital 
00029 HUS 
 
 
Department of Intensive Care Medicine 
Kuopio University Hospital 
Kuopio 
 
Department of Intensive Care Medicine 
Tampere University Hospital 
Tampere 
 
Department of Intensive Care Medicine 
Turku University Hospital 
 
Methodological trial sites 
Coordinating centre 
Centre of Research in Intensive Care – CRIC 
Rigshospitalet 
Tagensvej 22 
2200 Copenhagen N 
+453545 7167 
contact@cric.nu 
 
Methods centre 
Copenhagen Trial Unit, Centre for Clinical Intervention Research 
Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen Ø 
 
 

 
 
 
 
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Statistical centre 
Section of Biostatistics 
University of Copenhagen 
Øster Farimagsgade 5 
1014 Copenhagen K 
 
Investigators 
Coordinating investigator 
Tine S Meyhoff, MD 
Dept. of Intensive Care 
Rigshospitalet 
Blegdamsvej 9 
2100 Copenhagen Ø 
+45 35450606 
classic@cric.nu 
 
National investigator for Denmark 
Anders Perner, senior staff specialist and professor 
Dept. of Intensive Care 
Rigshospitalet 
Blegdamsvej 9 
DK-2100 Copenhagen 
anders.perner@regionh.dk 
 
National investigator for Finland 
Ville Pettilä, Director, Professor 
Division of Intensive Care Medicine, 
Department of Anaesthesiology, Intensive Care and Pain Medicine, 
University of Helsinki and Helsinki University Hospital 
Haartmaninkatu 4, 
00029 HUS, Finalnd 
ville.pettila@hus.fi 
 
National investigator for Sweden 
Maria Cronhjort, PhD, ICU Consultant 
Karolinska Institutet, Dept. of Clinical Science and Education, Section of Anaesthesia and Intensive 
Care 
Södersjukhuset 
Sjukhusbacken 10 
11883 Stockholm 
Sweden 
+46 8616 2965 
maria.cronhjort@sll.se 
 
National investigator for Norway 
Jon Henrik Laake, PhD, Senior Staff Specialist 
Department of Anaesthesia and Intensive Care Medicine 
Rikshospitalet, Oslo University Hospital 

 
 
 
 
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Sognsvannsveien 20 
0372 Oslo 
Norway 
jon.henrik.laake@rikshospitalet.no 
 
 
National investigator for Holland 
Iwan van der Horst 
Department of Intensive Care 
University Medical Center Groningen 
 
9713 Groningen 
The Netherlandsi.c.c.van.der.horst@umcg.nl 
 
National investigator for France 
Michael Darmon, PhD, Senior Staff Specialist 
Department of Intensive Care Medicine 
Hopital St. Louis 
Avenue Claude Vellefaux 
75010 Paris 
France 
Michael.darmon@aphp.fr 
 
National investigator for Czech 
Marek Nalos, Senior Staff Specialist 
Medical Intensive Care Unit (MJIP) 
1. Interni klinika 
Fakultni Nemocnice 
Alej Svobody 80 
Plzen 30460 
Czech Republic  
mareknalos@gmail.com 
 
National investigator for the UK 
Marlies Ostermann 
Guy’s and St Thomas’ Hospital 
Westminster Bridge Road 
London SE1 7EH 
Marlies.Ostermann@gstt.nhs.uk 
 
National investigator for Spain 
Dr. Ricardo Ferrer Roca 
Dept. of Intensive Care 
Hospital Vall d'Hebron 
Passeig de la Vall d'Hebron, 119-129 
08035 Barcelona 
Spain 
r.ferrer@vhebron.net 
 
National Investigator for Switzerland 
Stephan M. Jakob, chief physician and professor 

 
 
 
 
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Dept. of Intensive Care Medicine 
University Hospital Bern (Inselspital) 
Freiburgstrasse 10 
CH-3010 Bern 
stephan.jakob@insel.ch 
 
National investigator for Italy 
Maurizio Cecconi, senior staff specialist 
Dept. of Intensive Care Medicine 
Humanitas Research Hospital, 
20089 Rozzano MI 
doctor.cecconi@gmail.com 
 
National investigator for Belgium 
Manu L.N.G. Malbrain, senior staff specialist 
Dept. of Intensive Care Medicine 
University Hospital Brussels (UZB)  
Laarbeeklaan 101  
1090 Brussel 
Manu.Malbrain@uzbrussel.be 
 
Site investigators in Denmark 
 
Anders Perner, senior staff specialist and professor 
Dept. of Intensive Care 
Rigshospitalet 
Blegdamsvej 9 
DK-2100 Copenhagen 
anders.perner@regionh.dk 
 
Søren Hoffmann, staff specialist 
Dept. of Anaesthesia and Intensive Care 
Bispebjerg Hospital, University of Copenhagen 
2200 Copenhagen N 
soeren.kristen.lundgaard.hoffmann.01@regionh.dk 
 
Pending 
Dept. of Anaesthesia and Intensive Care 
Herlev Hospital, University of Copenhagen 
2730 Herlev 
 
Morten Bestle, senior staff specialist and associate professor 
Dept. of Anaesthesia and Intensive Care 
North Zealand Hospital, University of Copenhagen 
3400 Hillerød 
morten.bestle@regionh.dk 
 
Louise Gyldensted, senior staff specialist 
Intensive Care Unit Q-022GE  

 
 
 
 
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Gentofte Hospital 
2900 Hellerup 
louise.gyldensted.01@regionh.dk 
 
Lars Nebrich, senior staff specialist 
Dept. of Anaesthesia and Intensive Care 
Zealand University Hospital, Køge 
4600 Køge 
lnec@regionsjaelland.dk 
 
Lene Russell, senior staff specialist 
Dept. of Anaesthesia and Intensive Care 
Zealand University Hospital, Roskilde 
4000 Roskilde 
lenru@regionsjaelland.dk 
 
Klaus Vennick Marcussen, staff specialist 
Dept. of Anaesthesia and Intensive Care 
Slagelse Hospital 
4200 Slagelse 
klvm@regionsjaelland.dk 
 
Marianne Vang, senior staff specialist 
Dept. of Anaesthesia and Intensive Care 
Randers Hospital 
8930 Randers 
marivang@rm.dk 
 
Michael Lindhardt Rasmussen, staff specialist 
Dept. of Anaesthesia and Intensive Care 
Herning Hospital 
7400 Herning 
Michael.Lindhardt.Rasmussen@vest.rm.dk 
 
Christoffer Sølling, senior staff specialist  
Dept. of Anaesthesia and Intensive Care 
Viborg Hospital 
8800 Viborg 
chrsoell@rm.dk 
 
Bodil S Rasmussen, senior staff specialist, professor 
Dept. of Anaesthesia and Intensive Care 
Aalborg University Hospital 
9000 Aalborg 
bodil.steen.rasmussen@rn.dk 
 
Anne Craveiro Brøchner 
Dept. of Anaesthesia and Intensive Care 
Lillebælt Hospital 
6000 Kolding 

 
 
 
 
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Anne.Craveiro.Broechner@rsyd.dk 
 
Site investigators in Finland 
 
Suvi Vaara, staff specialist, associate professor 
Division of Intensive Care Medicine, 
Department of Anaesthesiology, Intensive Care and Pain Medicine, 
University of Helsinki and Helsinki University Hospital 
Haartmaninkatu 4, 
00029 HUS, Finalnd 
suvi.vaara@hus.fi 
 
 
Stepani Bendel, senior staff specialist, associate professor 
Department of Intensive Care Medicine 
Kuopio University Hospital 
stepani.bendel@kuh.fi 
 
Anne Kuitunen, senior staff specialist, associate professor 
Tampere University Hospital 
Department of Intensive Care Medicine 
anne.kuitunen@pshp.fi 
 
Juha Grönlund, staff specialist, associate professor 
Turku University Hospital 
Department of Intensive Care Medicine 
juha.gronlund@tyks.fi 
 
 
Independent Data Monitoring and Safety Committee (IDMSC)  
Pending. 
 
 
 
Signatures 
 
Anders Perner, sponsor 

 
 
 
 
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Table of contents 
 
Abstract ............................................................................................................ 2 
Administrative information ............................................................................. 3 
List of abbreviations...................................................................................... 13 
1 
Introduction and background ................................................................ 15 
1.1 
The participant population - patients with septic shock ..........................................15 
1.2 
Current practice in septic shock ............................................................................15 
1.3 
Trial interventions – restrictive vs. standard IV fluid therapy ..................................16 
1.4 
Risks and benefits .................................................................................................19 
1.5 
Ethical justification and trial rationale ....................................................................19 
1.6 
Trial conduct .........................................................................................................20 
2 
Trial objectives ........................................................................................ 21 
3 
Trial design .............................................................................................. 21 
3.1 
Trial design ...........................................................................................................21 
3.2 
Randomisation ......................................................................................................21 
3.3 
Blinding .................................................................................................................21 
3.4 
Participant timeline ................................................................................................21 
3.5 
End of trial .............................................................................................................22 
4 
Selection of participants ........................................................................ 22 
4.1 
Inclusion criteria ....................................................................................................22 
4.2 
Exclusion criteria ...................................................................................................22 
4.3 
Participant discontinuation and withdrawal ............................................................23 
4.3.1 
Discontinuation and withdrawal at the choice of the participant or the proxy .....23 
4.3.2 
Discontinuation and withdrawal at the choice of the investigator .......................23 
4.3.3 
Discharge to another ICU ..................................................................................23 
5 
Selection of trial sites and personnel ................................................... 24 
5.1 
Trial sites and setting ............................................................................................24 
5.2 
Trial personnel ......................................................................................................24 
6 
Trial interventions ................................................................................... 24 
6.1 
Experimental intervention ......................................................................................24 
6.2 
Control intervention ...............................................................................................24 
6.3 
Co-interventions ....................................................................................................25 
6.4 
Concomitant interventions .....................................................................................25 
6.5 
Criteria for modification of interventions for a given trial participant .......................25 
6.6 
Assessment of participant compliance ..................................................................25 
7 
Outcome measures ................................................................................. 26 
7.1 
Primary outcome ...................................................................................................26 
7.2 
Secondary outcomes ............................................................................................26 
8 
Safety ....................................................................................................... 26 
8.1 
Definitions .............................................................................................................26 
8.2 
Risk and safety issues in the CLASSIC trial ..........................................................27 

 
 
 
 
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8.3 
Assessment of adverse events .............................................................................27 
8.3.1 
Timing ...............................................................................................................27 
8.3.2 
Classification of an event ..................................................................................27 
8.4 
Reporting ..............................................................................................................27 
9 
Procedures, assessments and data collection .................................... 28 
9.1.1 
Screening ..........................................................................................................28 
9.1.2 
Procedures for informed consent ......................................................................28 
9.2 
Data collection ......................................................................................................28 
9.2.1 
Methods ............................................................................................................28 
9.2.2 
Timing ...............................................................................................................28 
9.3 
Data management ................................................................................................31 
9.4 
Confidentiality .......................................................................................................31 
9.5 
Collection, handling, storage and transportation of human biological material .......31 
9.6 
Access to data ......................................................................................................31 
10 
Statistical plan and data analysis ...................................................... 31 
10.1 
Sample size and power .........................................................................................31 
10.1.1 
Sample size estimation .................................................................................31 
10.1.2 
Power estimations .........................................................................................32 
10.2 
Statistical methods ................................................................................................32 
10.2.1 
Significance ...................................................................................................33 
10.2.2 
Interim analysis .............................................................................................33 
10.2.3 
Early stopping criteria ....................................................................................33 
10.2.4 
Accountability procedure for missing data/population for analysis .................34 
11 
Quality control and quality assurance .............................................. 34 
11.1 
Monitoring .............................................................................................................35 
11.2 
Drug traceability measures ...................................................................................35 
12 
Legal and organisational aspects ...................................................... 35 
12.1 
Finance .................................................................................................................35 
12.1.1 
Trial funding ..................................................................................................35 
12.1.2 
Compensation ...............................................................................................35 
12.2 
Insurance ..............................................................................................................35 
12.3 
Plan for publication, authorship and dissemination ................................................35 
12.4 
Sub-studies ...........................................................................................................36 
12.5 
Intellectual property rights .....................................................................................36 
12.6 
Organisational framework .....................................................................................36 
12.7 
Trial timeline .........................................................................................................37 
13 
References ........................................................................................... 37 
14 
Appendix .............................................................................................. 43 
14.1 
Appendix 1.  Research programme organisation...................................................44 
14.2 
Appendix 2. Trial definitions ..................................................................................45 
    14.2.1  Pre-screening protocol for Italian sites .............................................................51 
14.3 
Appendix 3. Adverse reactions not registered in CLASSIC ...................................53 
14.4 
Appendix 4. Charter for the independent data monitoring and safety committee ...54 

 
 
 
 
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14.5 
Appendix 5. Informed consent, Denmark ..............................................................60 
    14.5.1            Informed consent, Sweden ......................................................................61 
14.6 
Appendix 6. Simplified Mortality Score for the Intensive Care Unit (SMS-ICU) ......62 
14.7 
Appendix 7. Co-enrolment ....................................................................................63 
14.7.1 
Co-enrolment agreement form ......................................................................65 
14.8 
Appendix 8. List of proposed sub-studies ..............................................................66 
    14.8.1      Approved substudy 
protocols…………………..………………………………...665 14.9.... Appendix 9. Preliminary 
results of our systematic review ........................................................................................76 
14.10    Appendix 10. Trial flow chart ................................................................................77 
14.11    Appendix 11. International Committee of Medical Journal Editors (ICMJE) form for 
potential conflict of interest ...............................................................................................78 
14.12    Appendix 12. LOG of protocol changes ................................................................81 
 

 
 
 
 
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List of abbreviations 
 
6S, Scandinavian Starch for Severe Sepsis/Septic Shock 
ACE, Angiotensin-Converting-Enzyme 
ANG-II, Angiotensin-II 
ALL, Acute Lymphoblastic Leukaemia 
AML,  Acute Myelogenous Leukaemia  
ARDS, Acute Respiratory Distress Syndrome 
B-PLL, B-cell prolymphocytic leukaemia 
CI, Confidence Interval 
CLASSIC, The Conservative vs. Liberal Approach to fluid therapy of Septic Shock in Intensive Care Trial 
CLL, Chronic Lymphocytic Leukaemia 
CML, Chronic Myelogenous Leukaemia  
CPAP, Continuous Positive Airway Pressure  
CRIC, Centre for Research in Intensive Care 
CT, Computed Tomography  
CTU, Copenhagen Trial Unit 
DMSC, Data Monitoring and Safety Committee 
ECG, Electrocardiography  
eCRF, electronic Case Report Form 
EHOSS-1, The “Early haemodynamic optimization using reload dependence during septic shock” trial 
EQ-5D, Euro Qual-5D 
EudraCT, European Union Drug Regulating Authorities Clinical Trial 
FEAST, Fluid Expansion as Supportive Therapy trial 
GI, Gastrointestinal  
GRADE, Grading of Recommendations Assessment, Development and Evaluation 
hCG, human Chorionic Gonadotropin 
HCL, Hairy Cell Leukaemia 
HRQol, health-related quality of life 
ICH-GCP, International Conference on Harmonisation on Good-Clinical -Practice 
ICMJE, International Committee of Medical Journal Editors 
ICU, Intensive Care Unit 
IQR, Interquartile Range  
ITT, intention-to-treat  
IV, Intravenous  
KDIGO, Kidney Disease Improving Global Outcomes 
LVEF, Left Ventricle Ejection Fraction  
MC, Management Committee 
MCAR, Missing Completely At Random 
MDRD, Modification of Diet in Renal Disease formula 
MNAR, Missing Not At Random 
MoCa, Montreal Cognitive assessment 
MoCa MINI, Montreal Cognitive assessment ‘5-miniute protocol’ 

 
 
 
 
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MRI, Magnetic Resonance Imaging 
NaCl, Sodium Chloride  
NYHA, New York Heart Association 
PTLD, post-transplant lymphoproliferative disorder 
RRI, Relative Risk Increase  
RRR, Relative Risk Reduction 
SAE, Serious Adverse Event 
SAR, Serious Adverse Reaction  
SBE, Standard Base Excess  
SD, Standard Deviation  
SLL, Small Lymphocytic Lymphoma 
SMS-ICU score, Simplified Mortality Score for the Intensive Care Unit 
SSC, Surviving Sepsis Campaign 
SSSP-1 and SSSP-2, Simple Septic Shock Protocol 
TARTARE 2S, Targeted Tissue Perfusion versus Macrocirculation-guided Standard Care in Patients 
with Septic Shock trial 
TFM, Targeted Fluid Minimisation trial 
T-PLL, T-cell prolymphocytic leukaemia 
TRISS, Transfusion Requirements in Septic Shock trial 
GLM, Generalised Linear Model 
WHO, World Health Organisation 

 
 
 
 
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1 
Introduction and background 
1.1 
The participant population - patients with septic shock 
Septic shock is common and often lethal [1]. The WHO estimates that 6.000.000 patients die of sepsis 
every year and have declared sepsis a global health priority [1]. Patients with septic shock have 90-
day mortality rates around 40% to 50% even in the developed part of the world [2, 3, 4, 5]. Many 
survivors get readmitted to hospital and have impaired health-related quality of life (HRQoL) [6] and 
struggle with long-term reduced cognitive function [7]. Sepsis care is, therefore, associated with high 
use of resources in the developed part of the world [8]. 
 
Septic shock is defined as the need for vasopressor therapy and elevated lactate levels related to an 
infection and most often other organ systems are also impaired, including the brain, lungs and 
kidneys [9]. The most frequent sources of infection in patients with septic shock are pneumonia and 
abdominal, urinary tract, and soft tissue infections [10, 2, 3, 4]. 
 
Taken together, any improvement in sepsis care will result in reduced suffering among patients and 
relatives and improved global health [1]. 
1.2 
Current practice in septic shock 
The mainstay therapy for initial management of patients with septic shock includes intravenous (IV) 
antibiotics and fluids, source control, and supportive care [11]. In the developed parts of the world, 
most patients with septic shock are cared for in intensive care units (ICUs) because of the need for 
continuous monitoring and the use of life-support including vasopressor therapy in most patients, 
mechanical ventilation in many, and renal replacement therapy in some patients [11]. Patient 
location before ICU admission for septic shock appear to differ between continents [10], but in 
Scandinavia approximately half of the patients are admitted from hospital wards and one quarter 
each from emergency departments and operating rooms [2, 3, 5]. 
 
The international Surviving Sepsis Campaign (SSC) guideline [11], which is supported by 25 medical 
societies, recommends that a fixed IV crystalloid fluid volume of 30 ml/kg is given in the first 3 hours, 
but this is based on low-quality evidence. The guidance for continued fluid therapy is to give IV fluid 
as long as circulatory parameters improve, but this also is not supported by firm evidence [11]. Thus, 
the guideline promotes higher IV fluid volumes merely on physiological grounds.  
 
While the volumes of IV fluid given to patients with septic shock in emergency departments before 
ICU admission may be less variable [12], the fluid volumes given in ICU vary between ICUs beyond 
what may be explained by differences in patient mix (Fig 1). 
 
 
 

 
 
 
 
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Fig 1. Median (IQR) IV fluid volumes given for resuscitation of patients with septic shock in 
Scandinavian ICUs participating in a fluid trial [5]. Unadjusted data are shown (the dashed horizontal 
line denotes the median of all patients), but variation remained after adjustment for important 
patient characteristics as indicated by the p-value*. The figure is copied from [5]. 
 
On the other hand, the types of IV fluids used vary less; crystalloid solutions are used for 
resuscitation in the majority of ICU patients including those with sepsis [13]. This is in line with the 
strong recommendation in the SSC guideline to use crystalloid solutions, either saline or buffered 
solutions, for the resuscitation in patients with septic shock [11]. The guideline recommends against 
the use of the synthetic colloid solutions (starches and gelatines) and suggests that albumin be 
considered only if high fluid volumes are given [11]. A restrictive transfusion strategy of blood 
products is recommended by the SSC guideline and noradrenalin is recommended as first line 
vasopressor [11]. 
 
1.3 
Trial interventions – restrictive vs. standard IV fluid therapy 
In the present CLASSIC trial, the protocol restricting IV fluids is the experimental intervention and the 
protocol aiming at standard care is the control intervention. 
 
Observational data 
A systematic review of mainly observational studies associated more positive fluid balances with 
increased mortality [14]. Also earlier administration of the initial 30 ml/kg of IV fluid was not 
associated with reduced mortality in a large cohort of sepsis patients in the US [15]; in contrast 
earlier administration of antibiotics was associated with reduced mortality [15]. As noted by the 
authors of the latter report, confounding by indication is, however, very difficult to control for in 
observational studies of IV fluids in patients with septic shock. 
 
Randomised trials in septic shock 
We are presently conducting a comprehensive systematic review of lower vs. higher IV fluid volumes 
for resuscitation of adult patients with septic shock using Cochrane methodology, GRADE, and Trial 

 
 
 
 
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Sequential Analysis in order to inform methodology of the trial including the sample size estimation 
[16]. In our search, we have only found 5 randomised trials, in which a difference in fluid volumes 
was the main aim between intervention groups (Table 1). 
 
One was our own multicentre, pilot CLASSIC trial [5], in which the protocol restricting IV fluid 
volumes resulted in lower fluid volumes given as compared to a protocol aiming at standard care. 
Thus, the trial showed that resuscitation fluids could be restricted in patients with septic shock in 9 
general ICUs in Scandinavia. Moreover, the exploratory outcome measures suggested benefit from 
fluid restriction including less kidney impairment [5]. 
 
The second trial was a single-centre pilot trial from an ICU in the US, in which IV fluid therapy was 
guided by daily assessments of fluid responsiveness vs standard care [17]. Less IV fluid may have 
been given in the intervention group at day 5, but this was not statistically significant; neither were 
any of the clinical outcomes. 
 
The third trial was a single-centre pilot trial from an ICU in France, in which IV fluid therapy was 
guided by fluid responsiveness (intervention) vs central venous pressure (control) aiming at reducing 
fluid volumes [18]. Less IV fluid was given per day in the intervention group, but total intravascular 
volume expansion may not have differed between groups (Table 1) and none of the clinical outcomes 
differed.  
 
The fourth and fifth trials were single-centre trials done in patients with sepsis in an emergency 
department in Zambia [19, 20]. The first of these trials was stopped early because all 8 patients with 
severe hypoxic respiratory failure allocated to the more liberal fluid protocol died [19]. In the second 
of the trials, patients with severe hypoxic respiratory failure were excluded [20]. Patients with septic 
shock were randomised to a simple resuscitation protocol including fixed volume IV fluid with the 
addition of vasopressor in case of persistent hypotension and blood transfusion in case of severe 
anaemia vs. treatment according to usual care in that setting. The main difference in the 
management between the two groups was that higher volumes of IV fluid were given at 6-hours in 
the protocolised care vs. usual care group. Mortality at hospital discharge and 28-days was higher in 
the protocolised vs. usual care group. More patients in the protocolised vs. usual care group 
experienced worsening of respiratory failure (36% vs. 22%), but mechanical ventilation was not 
available for the trial patients, which reduces the generalisability of the results in a developed world 
setting.  
 
 

 
 
 
 
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Table 1. Randomised trials of fluid resuscitation of adult patients with septic shock, in which a 
strategy was used to obtain differences in fluid volumes between intervention groups 
Trial 
Setting 
Patients 
Median IV fluid 
volumes* 
Mortality** 
The CLASSIC pilot 
trial [5] 
9 Scandinavian 
ICUs 
153 patients with 
septic shock who 
had received 30 
ml/kg of IV fluid  
Lower fluid group 
0.5 L 
Higher fluid 
group 
2.0 L 
Lower fluid group 
33% 
Higher fluid 
group 
41% 
Targeted fluid 
minimisation (TFM) 
trial [21] 
Single US ICU 
82 patients with 
septic shock 
using vasopressor 
>12 h after initial 
resuscitation  
Lower fluid group 
6.2 L 
Higher fluid 
group 
8.7 L 
Lower fluid group 
56% 
Higher fluid 
group 
49% 
Early 
haemodynamic 
optimization using 
reload dependence 
during septic shock 
(EHOSS-1) trial [22] 
Single French 
ICU 
61 patients with 
septic shock who 
had received 25 
ml/kg of IV fluid 
Lower fluid group 
3.0 L 
Higher fluid 
group 
3.3 L 
Lower fluid group 
23% 
Higher fluid 
group 
47% 
Simple septic shock 
protocol (SSSP)-1 
[19] 
An emergency 
department in 
Zambia 
120 patients with 
suspected 
infection, 2 
positive SIRS 
criteria and organ 
dysfunction 
Lower fluid group 
1.6 L 
Higher fluid 
group 
2.9 L 
Lower fluid group 
61% 
Higher fluid 
group 
64% 
SSSP-2 [20] 
An emergency 
department in 
Zambia 
212 patients with 
suspected or 
proven infection 
and hypotension 
Lower fluid group 
2.0 L 
Higher fluid 
group 
3.5 L 
Lower fluid group 
33% 
Higher fluid 
group 
48% 
* At 6-h in SSSP-1 and -2, at day 5 in CLASSIC and TMF and at end of study in EHOSS-1; in TMF all 
fluids were recorded, in the other trials only resuscitation fluids were recorded 
**In-hospital in SSSP-1 and -2 and TMF, day 28 in EHOSS-1 and day 90 in CLASSIC 
 
Regarding the period after resuscitation of patients with sepsis, a systematic review of randomised 
trials indicated a 14% relative (7% absolute) risk reduction in mortality with more restrictive vs. more 
liberal fluid strategies, but less than 400 patients were included in the sepsis subgroup and the result 
was not statistically significant [23]. In the analysis of all patients with sepsis or acute respiratory 
distress syndrome (ARDS), a reduction in the time on mechanical ventilation was observed in the 
fluid restrictive vs. more liberal fluid strategy groups [23]. 
 
Randomised trials in other patient groups 
The results of two large randomised trials in other patient groups have favoured restriction of IV 
fluids as compared to more liberal approaches. A trial done in ICU patients with acute lung injury in 
the US indicated reduced time on mechanical ventilation and in ICU with restriction of IV fluid vs. 
standard care [24]. Furthermore, the FEAST trial in African children with infection and circulatory 

 
 
 
 
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impairment showed increased mortality in the children receiving IV fluid boluses as compared to 
those who did not receive fluid boluses [25]. 
 
Physiological data 
Restricting fluid therapy may be beneficial in reducing venous back-pressure and organ oedema, 
thereby improving organ function including that of the lungs, gut and kidneys [26, 27]. On the other 
hand, fluid restriction may also compromise peripheral and/or organ perfusion through reduced 
cardiac output and thereby reduced microcirculation from the arterial side [28]. Thus, the 
physiological data support the clinical equipoise and the need for large trials on IV fluid volumes in 
patients with septic shock. 
 
Following these notions, a recent consensus statement from key opinion leaders within the field of 
intensive care medicine gave the highest priority to trials on restrictive vs. liberal fluid therapy when 
prioritizing the topics that need testing in patients with septic shock [29]. 
 
1.4 
Risks and benefits 
The CLASSIC trial will be conducted in an ICU-setting where patients around the clock are monitored 
and cared for by ICU nurses and doctors who are trained to manage patients with circulatory failure 
including the titration of the different interventions used for these patients (i.e. IV fluids, 
vasopressors and inotropes). We therefore believe that it is safe for individual patients to be enrolled 
into the CLASSIC trial. 
 
As described above, clinical practice data, randomised trial data and those from observational and 
physiological studies do not provide firm evidence that one of the interventions in the CLASSIC trial is 
superior to the other, i.e. clinical practice variation and equipoise exists. We therefore believe that 
the CLASSIC trial is safe for the patients also at the group level. As the current guidelines promote 
higher fluid volumes and our hypothesis is that fluid restriction is beneficial, the CLASSIC trial patients 
may benefit from participation. 
1.5 
Ethical justification and trial rationale 
Septic shock carries a high risk of death and disability. Currently all patients with septic shock receive 
IV fluids, but the guidelines are based on low-quality evidence as no large trial has assessed the 
benefit vs. harm of lower vs. higher fluid volumes in these patients. The CLASSIC trial will be 
conducted to the highest of methodological standards assessing the benefit and harm of fluid 
restriction on patient-important outcome measures. Therefore, future patients will benefit from the 
CLASSIC trial results, regardless of the direction of the effect, as the results will enable better fluid 
therapy for septic shock. As outlined above, we believe that this can be done without additional risk 
for the patients enrolled into the trial. 
 
The patients to be enrolled in the CLASSIC trial cannot consent due to the combination of severe 
infection and circulatory shock. This will be applicable to the entire population of septic shock in the 

 
 
 
 
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ICU. No other patient groups may be investigated to improve IV fluid therapy in septic shock as no 
other groups have the combination of infection and shock.  
 
In addition, septic shock is a medical emergency that requires immediate interventions including fluid 
therapy [11]. Therefore, we cannot delay enrolment and need to use the consent procedures for 
emergency research. 
 
Consent will be obtained according to national law. In Denmark, we will use the consent procedures 
for temporarily incompetent patients for all patients 
(https://www.retsinformation.dk/Forms/R0710.aspx?id=192671).  The CLASSIC trial patients will be 
enrolled after informed consent from a doctor, who is independent of the trial, who has knowledge 
of the clinical condition and who is familiar with the trial protocol to such extent that he or she can 
judge for each patient if it will be reasonable to enrol the patient in the trial (the first trial guardian).  
In the CLASSIC trial, the first trial guardian will be named by the investigator at each Danish site 
before initiation of the trial. As soon as possible after enrolment, consent will be obtained from the 
patient’s next of kin and a second doctor (the second trial guardian). The second trial guardian must 
be different from the first trial guardian, but also independent of the trial. Patients, who regain 
competence, will be asked for informed consent as soon as possible (Appendix 5). The process 
leading to informed consent will be in compliance with all applicable regulations. The consenting 
subjects will be provided with written and oral information about the trial allowing them to make an 
informed decision about participation in the trial. Written information and the consent form will be 
subject to review and approval by the ethical committee system according to national law in all 
participating countries. The consenting party can at any time, without further explanation, withdraw 
consent. The process leading to informed consent may differ in the participating countries, but will 
be described and be in compliance with all applicable regulations in the country. 
  
1.6 
Trial conduct 
The CLASSIC trial will be conducted in compliance with the published trial protocol, the Helsinki 
Declaration in its latest version [30], the good clinical practice (GCP) guidelines [31], and national 
laws in the participating countries. We have written the protocol in accordance with the SPIRIT 2013 
Statement [32] and will register the trial in the www.clinicaltrials.gov and European Union Drug 
Regulating Authorities Clinical Trials (EudraCT) registries before the enrolment of the first participant. 
No substantial deviation from the protocol will be implemented without prior review and approval of 
the regulatory authorities except where it may be necessary to eliminate an immediate hazard to the 
trial participants. In such case, the deviation will be reported to the authorities as soon as possible. 
 
Enrolment will start after approval by the ethics committees, medicines agencies, data protection 
agencies and health authorities in the participating countries. We will publish the approved protocol 
at www.cric.nu and submit a manuscript with main points of the protocol including description of 
design, rationale and statistical analysis plan to a peer-reviewed medical journal. 
 

 
 
 
 
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2 
Trial objectives 
The objective of the CLASSIC trial is to assess benefits and harms of IV fluid restriction vs. standard of 
care on patient-important outcome measures in adult ICU patients with septic shock. 
 
We hypothesise that fluid restriction vs. standard care will improve patient-important outcome 
measures in septic shock.  
3 
Trial design 
3.1 
Trial design 
The CLASSIC trial is an investigator-initiated, international multi-centre, parallel-grouped, open-
labelled, centrally randomised, stratified, outcome assessor- and analyst-blinded trial with adequate 
generation of allocation sequence, and allocation concealment. 
3.2 
Randomisation 
Patients with septic shock fulfilling the inclusion criteria will be randomised if they do not fulfil an 
exclusion criterion. The 1:1 randomisation will be centralised and web-based according to the 
computer-generated allocation sequence list, stratification variables (haematological or metastatic 
cancer (Y/N) and trial site), and varying block size at Copenhagen Trial Unit (CTU). The allocation 
sequence list will exclusively be known to the data manager at CTU and will be unknown to the 
investigators to allow immediate and concealed allocation to one of the two intervention groups. 
Each participant will be allocated a unique patient-screening number. 
3.3 
Blinding 
Fluid restriction vs standard care fluid therapy cannot be blinded for investigators, clinical staff or 
participants. We will mask the allocation for the research staff who assess the long-term outcomes 
and for the trial statistician. Also, the management committee will write two abstracts based on the 
statistical report with the group allocation masked, one assuming the experimental intervention 
group is X and the control intervention group is Y, and one assuming the opposite. After this the 
allocation code will be unmasked [33, 34]. 
3.4 
Participant timeline  
We will strive to enrol patients as soon as they fulfil the inclusion criteria. The participants will 
continue the allocated intervention until they are discharged from the ICU with a maximum of 90 
days after randomisation.  If the participant is readmitted to the ICU within 90 days after 
randomisation, she/he will continue the allocated fluid therapy. 
 
We will follow the participants for 1-year after randomisation and identify 1-year survivors in 
hospital, regional or national registries. We will contact all 1-year survivors approximately 2 weeks 
after to assess health related quality of life and cognitive function by interview. 

 
 
 
 
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3.5 
End of trial 
The trial will end when the last patient enrolled has completed 1 year follow-up (last-patient-last 
visit). We will contact the last patient approximately 2 weeks after the 1-year follow-up date and 
allow 1 month of response time for the 1-year follow-up.  
We will report the end-of-trial no later than 90 after last patient last visit to the Danish Medicines 
Agency and Ethics Committee.  
  
4 
Selection of participants 
All patients admitted or planned to be admitted to an active trial site will be considered for 
participation. Patients will be eligible, if they comply with the inclusion and exclusion criteria below. 
We aim to include the patients as early as possible and exclude patients who may have specific fluid 
needs.  
4.1 
Inclusion criteria 
All the following criteria must be fulfilled: 
- 
Aged 18 years or above 
- 
Admitted to the ICU or plan to be admitted to the ICU regardless of trial participation 
- 
Septic shock defined according to the Sepsis-3 criteria [9]: 
o Suspected or confirmed site of infection or positive blood culture AND 
o Ongoing infusion of vasopressor/inotrope agent to maintain a mean arterial 
blood pressure of 65 mmHg or above AND 
o Lactate of 2 mmol/L or above in any plasma sample performed within the last 
3-hours 
- 
Have received at least 1 L of IV fluid (crystalloids, colloids or blood products) in the last 
24-hours prior to screening. 
4.2 
Exclusion criteria 
We will exclude patients who fulfil any of the following criteria: 
- 
Septic shock for more than 12 hours at the time of screening because we want to include 
patients early in their course 
- 
Life-threatening bleeding as these patients need specific fluid/blood product strategies 
- 
Acute burn injury of more than 10% of the body surface area as these patients need a 
specific fluid strategy 
- 
Known pregnancy (details presented in Appendix 2)  
- 
Consent not obtainable as per the model approved for the specific site. 
 
We will not exclude patients enrolled in other interventional trials unless the protocols of the two 
trials collide; we present the rational for this in Appendix 7. Co-enrolment agreements will be 
established with the sponsor/investigator to maintain an updated list of trials approved for co-
enrolment (Appendix 7). 

 
 
 
 
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4.3 
Participant discontinuation and withdrawal 
The procedure of handling withdrawal of consent from a participant will follow national regulations 
for each participating country. 
4.3.1 
Discontinuation and withdrawal at the choice of the participant or the 
proxy 
A participant, who no longer wishes to participate in the trial, can withdraw his/her consent at any 
time without need of further explanation, and without consequences for further treatment. For 
incompetent participants, consent can be withdrawn at any time by the person(s), who has given 
proxy-consent. To limit the amount of missing data, we will collect as much data as possible from 
each participant. Therefore, if possible, the investigator will ask the participant or the proxy to which 
extent the withdrawal includes: 
• 
receiving the trial intervention only (allowing for all data registration and follow-up) 
 
OR 
• 
receiving the trial intervention AND further registration of daily data and/or follow-up 
 
Only the participant can demand deletion of already registered data and only if the participant did 
not consent previously. If so, the data will be deleted, and a new participant will be enrolled to 
obtain the full sample size. 
4.3.2 
Discontinuation and withdrawal at the choice of the investigator 
A participant may have the intervention stopped by the clinician or investigator at any time, if: 
• The participant experiences intolerable adverse reactions or events (including SAR or SUSAR) 
suspected to be related to the trial intervention. 
• The clinicians in conjunction with the coordinating investigator decide it to be in the interest of 
the participant 
• The participant after inclusion is subject to involuntary hospitalization, the intervention will stop.  
In these cases, the collection of data and the follow-up will continue, and the participant will remain 
in the intention-to-treat population. 
4.3.3 
Discharge to another ICU 
Participants who are discharged to another ICU will be regarded as discharged from the ICU unless 
the receiving ICU is an active CLASSIC trial site. If so, the participant will continue the allocated 
intervention at the new trial site until discharge from ICU. Participants referred to intermediate or 
step-up/step-down beds cared for by ICU staff trained in the CLASSIC trial protocol will continue the 
allocated intervention. 
 

 
 
 
 
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5 
Selection of trial sites and personnel 
5.1 
Trial sites and setting 
Trial sites will be ICUs in Europe and potentially in Canada and Australasia where we explore the 
possibilities for collaboration. Trial sites are listed in the section Administrative information (p. 3). 
This section will be updated during the trial. 
5.2 
Trial personnel 
All doctors caring for patients in participating ICUs will be eligible to enrol patients in the trial and all 
clinicians caring for patients will be eligible to care for and perform the interventions in the trial 
participants. All participating ICUs will receive written and oral instructions about the trial 
procedures. A 24-hour hotline will be available for trial-related questions. 
6 
Trial interventions 
The intervention period is the entire ICU admission to a maximum of 90 days. 
6.1 
Experimental intervention 
No IV fluids should be given unless one of the below extenuating circumstances occurs; in these 
cases, IV fluid may be given in measured amounts: 
• In case of severe hypoperfusion or severe circulatory impairment defined by either: 
- Lactate 4 mmol/L or above 
- Mean arterial blood pressure below 50 mmHg (with or without vasopressor/inotrope) 
- Mottling beyond the kneecap (mottling score >2) OR  
- Urinary output less 0.1 mL/kg bodyweight/h, but only in the first 2 hrs after randomisation 
A bolus of 250-500 ml of IV crystalloid solution may be given followed by re-evaluation. These 
criteria identify patients at increased risk of death [35, 36, 37], and were found feasible and 
not associated with harm in the CLASSIC pilot trial [5]. 
 
• In case of overt fluid losses (e.g. vomiting, large aspirates, diarrhoea, drain losses, bleeding or 
ascites tap) IV fluid may be given to correct for the loss, but not above the volume lost. 
• In case the oral/enteral route for water or electrolyte solutions is contraindicated or has failed as 
judged by the clinical team, IV fluids may be given to:  
- Correct dehydration or electrolyte deficiencies. 
- Ensure a total fluid input of 1 L per 24 h (fluids with medications and nutrition counts as input). 
IV fluids may be given as carrier for medication, but the volume should be reduced to the lowest 
possible volume for the given medication. 
6.2 
Control intervention 
There will be no upper limit for the use of either IV or oral/enteral fluids. In particular: 

 
 
 
 
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• IV fluids should be given in the case of hypoperfusion or circulatory impairment and should be 
continued as long as hemodynamic variables improve including static or dynamic variable(s) as 
chosen by the clinicians. These criteria are based on the SSC guideline [11] 
• IV fluids should be given as maintenance if the ICU has a protocol recommending maintenance 
fluid 
• IV fluids should be given to substitute expected or observed loss, dehydration or electrolyte 
derangements 
6.3 
Co-interventions 
Types of fluid to be used in both intervention groups: 
• IV fluids given for circulatory impairment: Only isotonic crystalloids are to be used as per the 
Scandinavian guideline for fluid resuscitation [38] 
• Fluids given to substitute overt loss: Isotonic crystalloids are to be used. If large amounts of 
ascites are tapped, then human albumin may be used [39] 
• Fluids used for dehydration: Water or isotonic glucose should be used 
• Fluids used for electrolyte disturbances: Fluids should be chosen to substitute the specific 
deficiency, including water in the case of severe hypernatremia 
• Blood products are only to be used on specific indications including severe bleeding, severe 
anaemia and prophylactic in case of severe coagulopathy. 
6.4 
Concomitant interventions 
The use of concomitant interventions for septic shock should be based on the updated international 
sepsis guidelines [11]. In particular, we suggest the following to trial sites: 
 
• Relevant antibiotics and source control for the infection 
• Noradrenalin as vasopressor 
• Renal replacement therapy based on conservative criteria [40] (i.e. severe hyperkalaemia (p-K > 
6.0 mmol/L), severe metabolic acidosis (s-bicarbonate < 10 mmol/L and pH < 7.20), persistent 
kidney injury >72 h (oliguria/anuria or s-creatinine has not declined to 50% of the peak), or severe 
fluid overload combined with hypoxic respiratory failure (P/F-ratio < (26 kPa (200 mmHg)). 
6.5 
Criteria for modification of interventions for a given trial participant 
The clinical team may at any time violate the protocol if they find it to be in the best interest of the 
participant. We will have a CLASSIC trial hotline to enable discussion around the clock between the 
clinicians caring for trial participants and the CLASSIC trial team regarding protocol related issues.   
6.6 
Assessment of participant compliance 
We will monitor protocol compliance at the trial site through the electronic case report form (eCRF) 
and alert trial sites in the case of clear violation (central monitoring). In addition, the trial will be 
externally monitored according to the GCP directive and the monitoring plan (section 11).    
 

 
 
 
 
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7 
Outcome measures 
7.1 
Primary outcome 
All-cause mortality at day 90 after randomisation 
7.2 
Secondary outcomes 
- 
Number of participants with one or more serious adverse events (SAEs) in the ICU defined as 
ischaemic events (cerebral, cardiac, intestinal or limb ischaemia) or as a new episode of severe 
acute kidney injury (modified KDIGO3 [41]) 
- 
Number of participants with one or more serious adverse reactions (SARs) to IV crystalloids in 
the ICU as defined in section 8.2 
- 
Days alive at day 90 without life support (vasopressor / inotropic support, invasive mechanical 
ventilation or renal replacement therapy) 
- 
Days alive and out of hospital at day 90 
- 
All-cause mortality at 1-year after randomisation 
- 
HRQoL 1-year after randomisation measured using the EuroQoL (EQ)-5D-5L and EQ-VAS scores. 
Participants who have died will be assigned the lowest possible scores 
- 
Cognitive function 1-year after randomisation as assessed by the Montreal Cognitive 
Assessment (MoCa) score  
 
Several of the secondary outcomes above are composite outcomes. The single components of these 
will also be analysed and presented in a supplement to the primary publication. 
 
8 
Safety 
8.1 
Definitions  
In the CLASSIC trial, we will use the definitions bellow [42]. 
 
Adverse Event (AE): any undesirable medical event occurring to a participant during a clinical trial, 
which does not necessarily have a causal relationship with the intervention. 
 
Adverse Reaction (AR): any undesirable and unintended medical response related to the 
intervention occurring to a participant during a clinical trial. 
 
Serious Adverse Event (SAE): any adverse event that results in death, is life-threatening, requires 
hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability 
or incapacity, or is a congenital anomaly or birth defect. 
 
Serious Adverse Reaction (SAR): any adverse reaction that results in death, is life-threatening, 
requires hospitalisation or prolongation of existing hospitalisation, results in persistent or significant 
disability or incapacity, or is a congenital anomaly or birth defect. 
 

 
 
 
 
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Suspected Unexpected Serious Adverse Reaction (SUSAR): any suspected adverse reaction which is 
both serious and unexpected (the nature or severity of which is not consistent with the information 
available to date).  
8.2 
Risk and safety issues in the CLASSIC trial 
The trial participants will all be ICU patients for whom all adverse events and reactions are 
documented routinely in the patient health record (i.e. ICU notes, charges and laboratory reports).  
We will record in the eCRF the occurrence of SAEs and SARs on all trial days in ICU for all included 
patients and report SAEs and SARs as outcomes measures.   
 
Both interventions groups will receive IV crystalloids as part of the protocol. We have identified the 
SARs to these fluids in the Danish Summary of Product Characteristics (SPCs) as listed below. The 
adverse reactions to the crystalloid solution not registered in the CLASSIC trial are listed in Appendix 
3 including the reasoning.  
 
SARs to crystalloid solutions (isotonic saline and buffered solutions (Ringer’s lactate, Ringer’s acetate 
and PlasmaLyte™)) assessed in the CLASSIC trial 
 
- Generalised tonic-clonic seizures 
- Anaphylactic reactions 
- Central pontine myelinolysis  
- Severe hypernatremia (defined as p-Na > 159 mmol/L) 
- Severe hyperchloraemic acidosis (defined as pH < 7.15 AND p-chloride > 115 mmol/L) 
- Severe metabolic alkalosis (defined as pH > 7.59 AND SBE > 9 mmol/L) 
8.3 
Assessment of adverse events 
8.3.1 
Timing 
In all participants, we will assess the occurrence of SAEs and SARs on all trial days the participants 
spend in ICU to a maximum of 90 days. 
8.3.2 
Classification of an event 
We will make no inferences about a causal relationship between the intervention and the 
event/reaction of the SAEs (7.2) and SARs (8.2), but register the occurrence in the two groups and 
report them in the final report according to the definition given above. As for any other SAE not 
covered in section 7.2 and 8.2, the investigators will report them if they are adjudicated to be related 
to the trial intervention to Sponsor or his delegate within 24 hours. If such a SAE is deemed related to 
the intervention by Sponsor and the investigator, it will be considered a SUSAR and reported as such 
(section 8.4).   
8.4 
Reporting 
Any serious adverse reaction/event not covered in the secondary outcomes (defined in 7.2 and 8.2) 
adjudicated to be related to the trial intervention by the investigator, will be reported within 24 

 
 
 
 
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hours to the Sponsor or his delegate. If deemed a SUSAR by the Sponsor, he will report it to the 
Danish Medicine Agency within 7 days after the report of a life-threatening or fatal SUSAR. No later 
than 8 days after the reporting, the Sponsor will inform the Danish Medicines Agency of relevant 
information on the Sponsor’s and the investigator’s follow-up action to the life-threatening or fatal 
SUSAR. Any other SUSARs will be reported to the Danish Medicines Agency no later than 15 days 
from the time when the Sponsor is informed. 
 
Once a year, the Sponsor will submit a list of all SARs that have occurred at all sites during the trial 
period and a report on safety of the trial subjects to the Danish Medicines Agency.  
 
The Sponsor will notify the Danish Medicines Agency when the trial has been completed (no later 
than 90 days thereafter) and if earlier than planned, the reasons for stopping the trial.  
 
In addition, we will report all SAEs and SARs defined in 7.2 and 8.2 as outcome measures and all 
SUSARs in the final trial report and the results of the trial will be reported on EudraCT.  
 
9 
Procedures, assessments and data collection 
9.1.1 
Screening 
All patients admitted to, or planned for clinical reasons to be admitted to a participating ICU with 
septic shock will be eligible for screening. 
9.1.2 
Procedures for informed consent 
Participants will be enrolled after consent by proxy is obtained according to national regulations. The 
procedure for each participating country will be described; that for Danish participants is described in 
Appendix 5. The Swedish procedure for consent and specific Swedish regulations are described in 
Appendix 5.1. 
9.2 
Data collection 
9.2.1 
Methods 
Data will be obtained from the participant’s hospital files and national/regional/hospital registers 
(source data as defined per site, region and country) and by participant survey/interview and entered 
in the web-based eCRF by trial investigators or their delegates. For participants transferred from a 
trial ICU to a non-trial ICU, data related to the outcomes will be collected according to national 
practice e.g. investigator contact to the non-trial ICU or health care registers. 
9.2.2 
Timing 
All variables are defined in Appendix 2. 
Baseline variables:  
- 
Sex 
- 
Date of birth 

 
 
 
 
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- 
Date of admission to hospital  
- 
Date and time of admission to ICU 
- 
From where was the participant admitted to ICU 
o Emergency department or directly from the pre-hospital setting 
o Hospital ward 
o Operating or recovery room 
o Another ICU 
- 
Focus of infection: 
o Pneumonia 
o Gastrointestinal infection 
o Urinary tract infection 
o Skin or soft tissue infection 
o Other 
- 
Co-morbidities:  
o Active hematologic cancer 
o History of metastatic carcinoma 
o History of ischemic heart disease or heart failure 
o History of chronic hypertension 
o Chronic dialysis  
- 
Blood values, interventions and vital parameters: 
o Participant weight 
o Highest plasma lactate value within the last 3 hours of randomisation 
o Highest dose of noradrenaline within the last 3 hours of randomisation 
o Volumes of IV fluid within the last 24 hours of randomisation 
o Use of systemic corticosteroids in the last 24 hours of randomisation 
o Highest plasma creatinine value within the last 24 hours of randomisation 
o Use of acute renal replacement therapy in the last 3 days prior to randomisation 
o Habitual plasma creatinine value prior to current hospitalisation (mark estimated or 
measured) 
- 
Values for the Simplified Mortality Score (SMS score)[43] at ICU admission not covered by 
the above: 
o Lowest measured systolic blood pressure in the last 24 hours prior to randomisation 
o Respiratory support within the last 24 hours of randomisation (support during surgery 
excluded)   
 
 
Daily during ICU admission (day form):  
- 
Fluid input and output 
o Total volume and specific type of IV isotonic crystalloids (isotonic saline and buffered 
solutions: Ringer’s lactate, Ringer’s acetate and PlasmaLyte™) 
o Total volume of other IV fluids 
o Total volume of albumin  
o Total volume of fluids with medications 
o Total volume of fluids with nutrition 
o Total volume of blood products 

 
 
 
 
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o Urinary output on this day (ml) 
o Total volume of other losses on this day including drainage, aspirates, stools and 
bleeding 
- 
Did a major protocol violation occur on this day? 
- 
Use of infusion of vasopressor or inotrope on this day 
- 
Use of systemic corticosteroids on this day 
- 
Use of mechanical ventilation on this day 
- 
Use of renal replacement therapy on this day 
- 
Plasma concentration of creatinine 
- 
SAEs on this day (y/n for everyone) 
o Cardiac ischemic event 
o Cerebral ischemic event 
o Intestinal ischemic event 
o Limb ischemia 
- 
SARs on this day (y/n for everyone) 
o General tonic-clonic seizures 
o Anaphylactic reactions 
o Central pontine myelinolysis.  
o Severe hypernatremia 
o Severe hyperchloraemic acidosis 
o Severe metabolic alkalosis   
 
ICU discharge form: 
- 
Died in ICU 
- 
Discharged to the ward at the same or another hospital 
- 
Discharged to another ICU participating in CLASSIC 
- 
Discharged to another ICU not participating in CLASSIC 
o Number of days of infusion of vasopressor or inotrope in this ICU 
o Number of days of mechanical ventilation in this ICU 
o Number of days of renal replacement therapy in this ICU 
 
Follow-up 90 days after randomisation: 
- 
Death (y/n, if yes date of death) 
- 
Date of the last session of any renal replacement therapy 
- 
Date of discharge from hospital 
- 
Additional hospital admissions 
 
Follow-up 1 year after randomisation 
- 
Death (y/n, if yes date of death) 
- 
EQ-5D-5L and EQ-VAS scores 
- 
MoCa scores 
 

 
 
 
 
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9.3 
Data management 
The data manager at CTU or his/her delegate will construct and oversee the eCRF. He/she will, as the 
only person, have access to the randomisation list during trial. The eCRF and the trial database will be 
hosted at the server of CTU with appropriate back-up and security as per the GCP regulative.  
9.4 
Confidentiality 
Each participant will receive a unique trial identification number. Trial investigators will receive a 
personal username and passwords to access the randomisation system and the eCRF. Each site will 
only have access to site specific participant data. Data will be handled according to the National Data 
Protection Agency and protected by the Danish national laws ‘Loven om behandling af 
personoplysninger’ and ‘Sundhedsloven’.  
9.5 
Collection, handling, storage and transportation of human biological 
material 
No additional sampling of human material will be done in the main trial as data entry will rely on 
routine testing done in the clinical setting. In sub-studies, blood tests will likely be taken in addition 
to the routine clinical tests. If so, specific protocols will be submitted for approval, as described in 
section 12.4.     
9.6 
Access to data 
All original records (incl. consent forms, eCRFs, and relevant correspondences) will be archived for 15 
years. De-identified data will be made publicly available 9 months after the publication of the outcome 
data according to the recent ICMJE recommendations [69]. As it is for all CRIC trials, all trial-related 
documents will be public available at www.CRIC.nu including those of the site master file, the eCRF 
template, instructions, educational material etc. 
10 Statistical plan and data analysis 
The analyses will be done according to the principles stipulated in ICH-GCP guidelines [42] and the 
detailed statistical analysis plan, which will be published before the randomisation of the last 
participant. 
10.1 
Sample size and power 
10.1.1 
Sample size estimation 
By enrolling 1554 (2 x 777) participants, we can show a 15% relative risk reduction (7% absolute) in 
the restrictive group from an estimated 45% 90-day mortality in the standard care group (data from 
our previous CLASSIC pilot, TRISS and 6S trials [2, 3, 5], systematic reviews [23, 44] and a recent large 
cohort study [4]) at type 1 and 2 error levels of 5% and 20% (power=80%), respectively. 

 
 
 
 
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10.1.2 
Power estimations 
We expect to have the following statistical power for the secondary outcomes based on 2 x 777 
participants, a type 1 error level of 1% and a relative risk reduction of 15% in the experimental vs 
control group: 
- 
50% power for the number of participants with one or more SAEs (control event rate 25%) 
- 
10% power for the number of participants with one or more SARs (control event rate 5%) 
- 
80% power for the mortality at 1-year (control event rate 55%). 
 
The estimates of control event rates originate in data of previous septic shock trials [2, 3]. We expect 
the following outcomes to be highly skewed (non-normally distribution): Days alive without life 
support and out of hospital at day 90 and HRQoL and cognitive function at 1-year. The power 
estimations for these are, therefore, somewhat uncertain why we refrain from making these 
estimates. 
10.2 
Statistical methods 
The analyses will be done in the intention-to-treat (ITT) population defined as all randomised 
participants for whom there is consent for the use of data. We will perform the primary analyses 
adjusted for the stratification variables [45] and challenge the primary result in analyses adjusted for 
important baseline risk factors (co-morbidities, higher SMS score, and focus of infection (other foci 
vs. urinary tract infection)[43, 46] and use of corticosteroids, which may reduce the time in ICU and 
thus the time exposed to the protocol [47]) and analyses of subgroups (Table 2) and the per-protocol 
population being the ITT population except those having one or more major protocol violations as 
defined below (Table 3). If there is more than 5% missing data for outcomes and/or covariates, we 
will impute the missing data using multiple imputations as described below (10.2.4). 
 
All analyses will be 2-tailed and we will use Fisher’s exact test for unadjusted comparisons of 
dichotomized outcomes (mortality at 90-days and 1-year, SAEs and SARs) and logistic regressions for 
adjusted analyses of these outcomes. The remaining secondary outcomes are continuous measures; 
we expect that these are highly skewed (non-normally distribution), because of inflation of specific 
values such as zero for days alive outside hospital for all patients who die while at the ICU. We will 
use statistical methods that can accommodate this type of data; the precise models will be specified 
in the detailed statistical plan. 
 
Several of the secondary outcome measures are composite; we will also analyse each component of 
these outcomes as recommended [42]; the precise models will be specified in the detailed statistical 
plan. 
 
Table 2. Heterogeneity of the intervention effects on the primary outcome will be analysed in the 
following subgroups based on baseline characteristics 
 
Subgroup 
Definition 
Expected direction of 
the interaction 
Statistical test 
Participants who are 
mechanically 
ventilated  
Invasive mechanically 
ventilated (yes/no) 
Larger effect of fluid 
restriction in 
mechanically 
ventilated participants 
Test of interaction in 
the adjusted analysis 
described above;  P-
value 0.01 

 
 
 
 
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Participants with 
severe acute kidney 
injury 
KDIGO (creatinine) 
criteria of 2 or above 
[41] (yes/no) 
Larger effect of fluid 
restriction in acute 
kidney injury 
Test of interaction in 
the adjusted analysis 
described above; P-
value 0.01 
Participants with 
severe metabolic 
failure 
Plasma lactate level 
above 4 mmol/l 
(yes/no) 
Larger effect of fluid 
restriction in severe 
metabolic failure 
Test of interaction in 
the adjusted analysis 
described above; P-
value 0.01 
Participant weight 
Weight tertiles as 
observed in the 6S and 
TRISS trial cohorts 
combined [2, 3] 
Larger effect of fluid 
restriction with lower 
weight 
Test of interaction in 
the adjusted analysis 
described above; P-
value 0.01 
IV fluid volume given 
prior to randomisation 
30 ml/kg or more 
given (yes/no) 
Larger effect of fluid 
restriction with less 
fluid given 
Test of interaction in 
the adjusted analysis 
described above; P-
value 0.01 
 
 
Table 3. The definitions of major protocol violations, the exclusion of which will form the per-
protocol population 
  
Intervention group 
Major protocol violation definition 
IV fluid restriction 
IV fluids given without one of the extenuating 
circumstances occurring 
Standard of care 
No IV fluids given 
 
10.2.1 
Significance 
We will present the results as adjusted absolute and relative risk differences, computed using glm-
models with appropriate link functions and binomial error-distribution, with confidence intervals (CI) 
using 95% CIs for the analyses of primary outcome (P-value 0.05) and 99% CIs for those of the 
secondary outcomes (P-value 0.01) due the multiplicity of these.  
10.2.2 
Interim analysis 
We will conduct three interim-analyses; one when 10% of participants have been followed for 30-
days, one when 30% of participants have been followed for 30-days and one when 50% of 
participants have been followed for 90-days. The Data Monitoring and Safety Committee (DMSC) will 
analyse only the fluid volumes and protocol violations in the two intervention groups in the two 
interim analyses to ensure that separation is occurring. At the third interim analysis they will assess 
fluid volumes, protocol violations, the 90-day mortality and the rates of SAEs and SARs in the ICU in 
the two intervention groups as described in the charter (Appendix 4). The DMSC will submit their 
recommendations to the Management Committee, which make the final decision regarding the 
continuing, pausing or stopping of the trial as described in the DMSC charter.  
10.2.3 
Early stopping criteria 
The trial will not be stopped unless the cumulative Z-value for effect size supersedes the Lan DeMets 
group sequential monitoring boundary corresponding to a P-value for effect of less than 0.005 

 
 
 
 
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(approximately). The trial will not be stopped for futility as an intervention effect less than a 15% 
relative risk reduction may be clinically relevant. However, the DMSC can recommend pausing or 
stopping the trial if continued conduct of the trial clearly compromises participant safety. 
10.2.4 
Accountability procedure for missing data/population for analysis 
If less than 5% of data are missing for any primary or secondary outcome, a complete case analysis 
without imputation of missing values will be performed. If missing data are more than 5%, a 
statistician masked for the intervention will assess whether data are ‘missing completely at random’ 
(MCAR criterion) based on a rational assessment of the pattern of missing data [48]. Little’s test will 
be used if doubt remains [49]. If it is concluded that data are not MCAR, multiple imputation using 
chained equations will be performed by creating 10 input datasets under the assumption that the 
data are ‘missing at random’ (MAR criterion) [50, 51]. We will use outcomes and the most important 
baseline characteristics in the multiple imputations as will be outlined in the detailed statistical 
analysis plan. 
 
If multiple imputations are used, then the primary result of the trial will be based on these data. The 
unadjusted, non-imputed analysis will also be presented. If multiple imputation is used because of 
missing outcome data, we will use a best-worst worst-best case scenario as a sensitivity analysis to 
assess the potential impact of any pattern of missingness including that the data are ‘missing not at 
random’ (MNAR criterion). In the ‘best-worst-case’ scenario it is assumed that all participants lost to 
follow-up in the experimental group have had a beneficial outcome (e.g. have survived, had no SAE 
etc.); and all those with missing outcomes in the control group have had a harmful outcome (e.g. 
have not survived; have had a SAE etc.). Conversely, in the ‘worst-best-case’ scenario, it is assumed 
that all participants who were lost to follow up in the experimental group have had a harmful 
outcome; and that all those lost to follow-up in the control group have had a beneficial outcome. 
When continuous outcomes are used, a ‘beneficial outcome’ will be defined as the group mean plus 
two SD of the group mean or highest possible value whichever is smallest, and a ‘harmful outcome’ 
will be defined as the group mean minus two SD of the group mean or lowest possible value 
whichever is highest. 
11 Quality control and quality assurance 
The Sponsor and the coordinating investigator will be responsible for organizing the trial sites 
including education of local investigators, research nurses, and other trial site personnel before the 
initiation of the trial. This education will be continuously documented in the site master file. An 
annual investigator meeting will be planned.  
 
After initiation, trial site investigators will be responsible for all trial-related procedures at their site, 
including education of staff in trial-related procedures, recruitment and follow-up of participants and 
entry of data. Clinical staff at the trial sites will be responsible for the treatment of trial participants. 
 

 
 
 
 
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11.1 
Monitoring 
The trial will be externally monitored according to the GCP directive and the monitoring and data 
verification plan including the documentation of informed consent of trial participants. The 
monitoring and data verification plan will be developed together with the GCP unit of Copenhagen 
University Hospital and adhered to by the staff monitoring trial sites in all countries. In addition, we 
will use central monitoring of site through the eCRF including adherence to the protocol.  
11.2 
Drug traceability measures 
The volumes of IV fluid administered will be registered in the eCRF for every day the participant is in 
the ICU to a maximum of 90 days. The registration of the batch numbers and the expiry dates of the 
IV fluids and the identity of the clinician administering the fluid will be registered as per standard 
practice at the sites. These data will not be registered in the trial documents, but can be obtained by 
the Sponsor or the authorities if needed. We believe that this is a safe procedure because the IV 
fluids used in the CLASSIC trial has been in clinical use for many years and the safety of single doses 
cannot be questioned. The same procedure was approved by the Danish Medicines Agency in the 
CLASSIC pilot trial (EudraCT no. 2014-000902-37). 
 
12 Legal and organisational aspects 
12.1 
Finance 
12.1.1 
Trial funding 
The trial is funded by an unrestricted grant from the Novo Nordisk Foundation and Sofus Friis’ 
foundation. None of the funding organisations have been or will be involved in the design, conduct, 
analyses, or reporting of the trial nor will they have ownership of the data.  
12.1.2 
Compensation 
All trial sites will be paid DKK 3000 (400 EUR) for each participant with completed 1-year follow-up 
status to partly compensate for the increased workload regarding screening, consent, inclusion, data-
entry and follow-up. 
12.2 
Insurance 
In Denmark, the Patient Insurance Association insures all trial participants. Patient insurance will be 
ensured before initiating the trial in each participating country. We will use external funding for the 
costs of insurance. 
12.3 
Plan for publication, authorship and dissemination  
All trial results whether positive, negative or neutral will be published preferably in a peer-reviewed 
medical journal. Furthermore, the results will be published at the CRIC home page (www.cric.nu). We 
will adhere to the CONSORT statement including the accountability of all patients screened 
(Appendix 10). 

 
 
 
 
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Authorship will be granted according to the guidelines from the International Committee for Medical 
Journal Editors (ICMJE; http://www.icmje.org/recommendations/browse/roles-and-
responsibilities/defining-the-role-of-authors-and-contributors.html). 
 
The listing of authors will be as follows on the primary publication: TS Meyhoff will be first author, PB 
Hjortrup the second, J Wetterslev the third, the next authors will be the national investigators 
according to the number of included participants per country, then the other members of the 
Management Committee, the trial statistician and trial site investigators dependent on the number 
of included participants per site. A. Perner will be the last and corresponding author. 
 
The Management Committee will grant authorship depending on personal input as per the 
Vancouver definitions. If a trial site investigator is to gain authorship on the primary publication, the 
site has to include 25 participants or more. If a site includes 50 participants, 2 authorships may be 
granted, at 75 participants 3 authorships and so on. Investigators on sites including less than 25 
participants may be granted authorship on the long-term outcome publication if they contribute 
significantly as per the Vancouver definitions. 
 
The DMSC and investigators not qualifying for authorship will be acknowledged with their names 
under ‘the CLASSIC Trial investigators’ in an appendix to the final manuscript. 
The funding sources will be acknowledged, but they will have no influence on the data handling or 
analyses, the writing of the manuscript or the decision to publish.  
12.4 
Sub-studies 
Sub-studies are planned at selected sites and more will be encouraged as long as they do not hamper 
the completion of the main protocol and can be conducted after approval of the specific protocol by 
the Management Committee and the authorities. Thus, specific protocols for any sub-studies will be 
submitted to and approved by the relevant authorities and ethic committees before the 
commencement of such studies. In Appendix 8, the presently proposed sub-studies are listed. 
12.5 
Intellectual property rights 
The CLASSIC trial group owns the trial data. The Contract between the Sponsor and an investigator 
will be reviewed and approved by the Unit for Research and Innovation (Law and Contracts) of the 
Capital Region (https://www.regionh.dk/english/research-and-innovation/Pages/default.aspx) and 
by the Medical Director of Rigshospitalet, dr. Per Jørgensen. 
12.6 
Organisational framework  
The CLASSIC trial will be managed by the Management Committee and overseen by the CRIC Steering 
Committee (Appendix 1). The day-to-day running of the trial will be done by the Sponsor, the 
Coordinating Investigator and the CRIC office. 

 
 
 
 
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12.7 
Trial timeline  
November 2017 – January 2018: Finalisation of the protocol among Management Committee and 
national investigators 
January - March 2018: Approval of the protocol by the Danish Medicines Agency and the Ethics 
Committee of the Capital Region 
March - September 2018: Approvals in the other countries, building of the eCRF and recruitment and 
education of trial sites 
September 2018: First Danish participant enrolled 
October 2018: Commencement of inclusion in other countries 
January 2019: 1st interim analysis 
May 2019: 2nd interim analysis 
December 2019: 2nd interim analysis 
September 2020: Last participant enrolled 
January 2021: 90 day follow-up completed, database cleaned 
February 2021: Data analysis and submission of the primary report for publication 
September 2021: One year follow-up completed 
 
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50.  Schafer JL Graham JW (2002) Missing data: our view of the state of the art. Psychol Methods 
7: 147-177. 
 
51.  Vickers AJ Altman DG (2013) Statistics notes: missing outcomes in randomised trials. BMJ 
346: f3438. 
 
52.  Wong A, Nyenhuis D, Black SE, Law LS, Lo ES, Kwan PW, Au L, Chan AY, Wong LK, Nasreddine 
Z, Mok V (2015) Montreal Cognitive Assessment 5-minute protocol is a brief, valid, reliable, 
and feasible cognitive screen for telephone administration. Stroke 46: 1059-1064. 
 
53.  Myles PS, Williamson E, Oakley J, Forbes A (2014) Ethical and scientific considerations for 
patient enrollment into concurrent clinical trials. Trials 15: 470. 
 
54.  Laine C, Taichman DB, Mulrow C (2011) Trustworthy clinical guidelines. Ann Intern Med 154: 
774-775. 
 
55.  Qaseem A, Forland F, Macbeth F, Ollenschlager G, Phillips S, Van der Wees P (2012) 
Guidelines International Network: toward international standards for clinical practice 
guidelines. Ann Intern Med 156: 525-531. 
 
56.  Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P, Schunemann HJ (2008) 
GRADE: an emerging consensus on rating quality of evidence and strength of 
recommendations. BMJ 336: 924-926. 

 
 
 
 
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57.  Burgess E, Singhal N, Amin H, McMillan DD, Devrome H (2003) Consent for clinical research 
in the neonatal intensive care unit: a retrospective survey and a prospective study. Arch Dis 
Child Fetal Neonatal Ed 88: F280-F285. 
 
58.  Green S, Liu PY, O'Sullivan J (2002) Factorial design considerations. J Clin Oncol 20: 3424-
3430. 
 
59.  Faden RR, Beauchamp TL, Kass NE (2014) Informed consent, comparative effectiveness, and 
learning health care. N Engl J Med 370: 766-768. 
 
60.  Randolph AG (2009) The unique challenges of enrolling patients into multiple clinical trials. 
Crit Care Med 37: S107-S111. 
 
61.  Cook D, McDonald E, Smith O, Zytaruk N, Heels-Ansdell D, Watpool I, McArdle T, Matte A, 
Clarke F, Vallance S, Finfer S, Galt P, Crozier T, Fowler R, Arabi Y, Woolfe C, Orford N, Hall R, 
Adhikari NK, Ferland MC, Marshall J, Meade M (2013) Co-enrollment of critically ill patients 
into multiple studies: patterns, predictors and consequences. Crit Care 17: R1. 
 
62.  Cook DJ, Ferguson ND, Hand L, Austin P, Zhou Q, Adhikari NK, Danesh V, Arabi Y, Matte AL, 
Clarke FE, Mehta S, Smith O, Wise MP, Friedrich JO, Keenan SP, Hanna S, Meade MO (2015) 
Coenrollment in a randomized trial of high-frequency oscillation: prevalence, patterns, 
predictors, and outcomes. Crit Care Med 43: 328-338. 
 
 
 

 
 
 
 
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14 Appendix 
Appendix 1: Research programme organisation 
Appendix 2: Trial definitions 
Appendix 2.1: Pre-screening protocol for Italian sites 
Appendix 3: Adverse reactions not registered  
Appendix 4: Charter for the independent Data Monitoring and Safety Committee 
Appendix 5: Informed consent procedure in Denmark 
Appendix 5.1: Informed consent procedure in Sweden  
Appendix 6: Simplified Mortality Score 
Appendix 7: Co-enrolment agreement form 
Appendix 8: List of proposed sub-studies 
Appendix 9: Preliminary results of our systematic review on restrictive vs. standard of care/more liberal 
fluid resuscitation in patients with septic shock. 
Appendix 10: Trial participant flow chart  
Appendix 11: International Committee of Medical Journal Editors (ICMJE) form for potential conflict of 
interest 
Appendix 12: LOG of protocol changes 
 
 
 

 
 
 
 
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14.1 
Appendix 1.  Research programme organisation 
 
 
 
 

 
 
 
 
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14.2 
Appendix 2. Trial definitions 
 
Definition of stratification variables  
Site: all participating intensive care units (ICUs) will be assigned a number identifying the unit. 
 
Metastatic cancer or hematologic malignancy: 
- 
Metastatic cancer: proven metastasis by surgery, CT scan or any other method 
- 
Haematological malignancy includes any of the following: 
o Leukaemia: Acute lymphoblastic leukaemia (ALL), acute myelogenous leukaemia 
(AML), chronic myelogenous leukaemia (CML), chronic lymphocytic leukaemia (CLL). 
o Lymphoma: Hodgkin's disease, and Non-Hodgkin lymphoma (e.g. small lymphocytic 
lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma and mantle cell 
lymphoma) 
o Hairy cell leukaemia (HCL), marginal zone lymphoma, Burkitt's lymphoma, post-
transplant lymphoproliferative disorder (PTLD), T-cell prolymphocytic leukaemia (T-
PLL), B-cell prolymphocytic leukaemia (B-PLL), Waldenström's macroglobulinemia 
and other NK- or T-cell lymphomas 
o Multiple myeloma/plasma cell myeloma  
 
Definition of the inclusion criteria 
Age: the age of the participant in whole years at the time of randomisation. The age will be calculated 
from date of birth. 
 
Admitted to the ICU or plan to be admitted to the ICU: We will only recruit sites that have the status 
as an ICU. These may oversee beds defined as high-dependency, step-up or step-down beds. We will 
consider these as being part of the trial site ICU if staff trained in the protocol looks after the patients 
in these beds. The medical doctors at the site ICUs may enrol patients from other locations in the 
hospital (e.g. emergency departments, general wards or the recovery room) if the patient for clinical 
reasons is planned to be admitted to the ICU. 
 
Septic shock: We will define septic shock at the time of screening according to the Sepsis-3 criteria 
[9] i.e.: 
- 
Suspected or confirmed site of infection or positive blood culture AND 
- 
Ongoing infusion of vasopressor/inotrope agent (norepinephrine, epinephrine, 
phenylephrine, vasopressin analogues, angiotensin, dopamine, dobutamin, milrinone or 
levosemindan) to maintain a mean arterial blood pressure of 65 mmHg or above AND 
- 
Lactate of 2 mmol/L or above in any plasma sample performed within the last 3-hours prior 
to screening 
 
Have received at least 1 L of IV fluid in the last 24-hours prior to screening: We will count all 
crystalloids (isotonic saline, Ringer’s and Plasmalyte™ solutions) colloids (albumin 4, 5 or 20%, 
gelatine, hydroxyethyl starch and dextran solutions) and blood products (units or red cells, plasma or 

 
 
 
 
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platelets) the participant has received according to the source data within the last 24-hours 
independent of location (in- or pre-hospital) including fluids with IV medication and IV nutrition. 
Intraosseous fluid will be counted as IV.  
 
Definition of the exclusion criteria 
-Septic shock for more than 12 hours at the time of screening: Septic shock according to the Sepsis-3 
criteria [9] (definition given above) for more than 12 hours at the time of screening. We will use 
infection and infusion of vasopressor/inotrope agent for more than 12 hours as a cutoff, as the 
plasma lactate value may change during the course of treatment 
 
-Life-threatening bleeding: clinical bleeding needing transfusion of blood products as defined by the 
clinicians 
 
-Acute burn injury of more than 10% of the body surface area: burn injury leading to the present ICU 
admission. Patients with burn injury who are readmitted to the ICU or were initially care for in a 
general ward and admitted to the ICU for infection may be screened to enrolment. The latest 
documented estimate of the burn area will be used as these may be down-graded after the initial 
assessments. 
 
-Known pregnancy: women with known pregnancy based on clinical examination, the history or 
human chorionic gonadotropin (hCG). We will not demand negative hCG-status in all eligible fertile 
women, because (i) screening for enrolment has to be done within the time window (waiting for the 
hCG result will delay screening and result in fewer fertile women included), (ii) the pregnancy rate is 
very low in ICU patients with septic shock and (iii) trial participation will not endanger the woman or 
the foetus. The same procedure was approved by the Danish Medicines Agency in the TARTARE 2S 
trial (EudraCT no. 2015-005112-15), which has many similarities to the CLASSIC trial (ICU patients 
with septic shock randomized to protocolised titration of an approved medicine (noradrenalin). 
 
-Consent not obtainable according to national regulations: patients where the clinician or 
investigator is unable to obtain the necessary consent before inclusion of the patient according to 
the national regulations. 
 
Definition of baseline variables  
-Sex: the genotypic sex of the participant 
-Age: defined in inclusion criteria 
-Date of admission to hospital: the date of admission to the first hospital the participant was 
admitted to during the current hospital admission 
-Date and time of admission to the ICU (or high-dependency or step-up/step-down beds; see 
inclusion criteria): the date of admission to the first ICU the participant was admitted to during the 
current hospital admission 
-From where was the participant admitted to ICU 
o Emergency department or directly from the pre-hospital setting: 
Accident/Emergency/Casualty/Acute department in the same or another hospital or direct 
admission to the ICU by an ambulance service or similar 

 
 
 
 
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o Hospital ward: Any location in the same or another hospital not covered in the other 3 
categories 
o Operating or recovery room: including surgical theatre, endoscopy and angiography suite 
and any recovery facilities observing patients following invasive procedures. 
o 
Another ICU: either within the same or another hospital 
 
Focus of infection (documented or suspected): 
o Pulmonary: e.g. pneumonia, or empyema  
o Gastrointestinal infection: e.g. primary, secondary or tertiary peritonitis, abscess, 
cholangitis, cholecystitis, or invasive diarrhoeal disease 
o Urinary tract infection: e.g. urinary tract infection, or pyelonephritis  
o Skin or soft tissue infection: e.g. cellulitis, phlegmon, erysipelas, or fasciitis 
o Other: other infectious focus documented or suspected including meningitis, endocarditis, 
osteomyelitis, arthritis and bacteraemia   
 
-Co-morbidities, must have been present in the past medical history prior to ICU admission and are 
defined as follows:  
o Active hematologic cancer: defined in the stratification variables 
o History of metastatic carcinoma: defined in the stratification variables 
o History of ischemic heart disease or heart failure: previous myocardial infarction, invasive 
intervention for coronary artery disease, stable or unstable angina, NYHA class 3 or 4 or 
measured LVEF < 40%. 
o History of chronic hypertension: treatment at time of hospital admission with any 
antihypertensive agent e.g. diuretics, adrenergic receptor antagonists 
(alpha/beta/alpha+beta blockers), alpha-2 receptor agonists, calcium channel blockers, 
ACE-inhibitors, ANG-II receptor antagonists, aldosterone antagonists. 
o Chronic dialysis: use of renal replacement therapy at least once a week e.g. chronic 
haemodialysis or haemofiltration, peritoneal dialysis  
 
- Blood values, interventions and vital parameters: 
o Participant weight: measured or estimated in kg 
o Highest plasma lactate value within the last 3 hours of randomisation: in mmol/L 
o Highest dose of noradrenaline within the last 3 hours of randomisation: highest infusion 
rate in µg/kg/minVolumes of IV fluid within the last 24 hours of randomisation:  all 
crystalloids (any saline, NaHCO3-, Ringer’s and Plasmalyte™ solutions), colloids (albumin 4, 
5 or 20%, gelatine, hydroxyethyl starch and dextran solutions) and blood products (units of 
red cells, plasma or platelets) the participant has received within the last 24-hours 
independent of location (in- or pre-hospital) including fluids with IV medication and IV 
nutrition. Intraosseous fluid will be counted as IV 
o  
o Use of systemic (IV, IM or oral/per GI tube) corticosteroids in the last 24 hours of 
randomisation including any dose of hydrocortisone, methylprednisolone, dexamethasone 
or prednisolone 

 
 
 
 
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o Highest plasma creatinine value within the last 24 hours of randomisation: in µmol/L 
o Use of acute renal replacement therapy in the last 3 days prior to randomisation: any form 
of renal replacement therapy (e.g. dialysis, hemofiltration or hemo-diafiltration) at any rate 
in the last 72 hours, which has been initiated during the current hospitalisation (including 
any stay in another hospital immediately prior that in the site) 
o Habitual plasma creatinine value prior to current hospitalisation: estimated or measured in 
µmol/L. If no there are no values recorded in source data, we will estimate habitual plasma 
creatinine 
-The simplified mortality score (Appendix 6) is based on 7 variables obtained in the 24 h prior to 
randomisation of a patient into the trial [43]. The variables include:  
o Age: defined in inclusion criteria 
o Lowest systolic blood pressure: either invasive or non-invasive in mmHg. In case of cardiac 
arrest within the 24-h period ‘0’ will be registered. 
o Acute surgical admission: Surgery during current hospital admission that was added to the 
operating room schedule.  
o Hematologic malignancy or metastatic cancer: Defined in the stratification variables. 
o Vasopressors/inotropes: Use of continuous infusion of vasopressor or inotrope (defined in 
the inclusion criteria). 
o Respiratory support: Use of invasive or non-invasive mechanical ventilation including 
continuous mask CPAP or CPAP via tracheostomy within the last 24 hours prior to 
randomisation. Intermittent CPAP and high flow nasal cannula oxygen therapy are NOT 
considered as respiratory support 
o Renal replacement therapy: Use of acute or chronic intermittent or continuous renal 
replacement therapy. 
Definition of daily collected variables:  
-Fluid input and output in mL cumulated from the 24-h ICU charts 
o Total volume and specific type of IV isotonic crystalloids:  isotonic saline and buffered 
solutions: Ringer’s lactate, Ringer’s acetate and PlasmaLyte™ 
o Total volume of other IV fluids: e.g. <10% glucose, glucose-potassium, sodium-potassium-
chloride, half-saline 
o Total volume of albumin (4, 5 or 20% solutions combined) 
o Total volume of fluids with medications: both parenteral and enteral 
o Total volume of fluids with enteral and parenteral nutrition: e.g. enteral nutrition solutions 
and >10% glucose, protein or lipid solutions given parenterally or enterally  
o Total volume of non-nutritional enteral/oral fluids: e.g. water or soft drinks 
o Total volume of blood products: defined in inclusion criteria 
o Urinary output on this day 
o Any fluid volume removed during renal replacement therapy  
o Total volume of other losses on this day including drainage, aspirates, stools and bleeding 
 
-Major protocol violation on this day: 

 
 
 
 
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o Restrictive group: IV fluids given without one of the extenuating circumstances occurring on 
this day (y/n) 
o Standard care group: the violations (no IV fluid given) will be assessed from all the day form 
registrations regarding fluid input at the end of trial for each participant 
 
-Use of infusion of vasopressor or inotrope on this day: defined in the inclusion criteria 
 
- Use of systemic corticosteroids on this day: defined in the baseline data 
 
-Use of mechanical ventilation on this day: invasive mechanical ventilation as the use of positive 
pressure ventilation using a ventilator via a cuffed tube (oral, nasal or tracheostomy). CPAP is NOT 
mechanical ventilation. 
 
-Use of renal replacement therapy on this day: any form of renal replacement therapy (e.g. dialysis, 
hemofiltration or hemo-diafiltration) at any rate on this day. 
 
-Plasma concentration of creatinine in µmol/L on this day 
 
Definition of outcome measures 
Primary outcome:  
90 day mortality: death from any cause within 90 days post-randomisation.  
 
Secondary outcomes:  
-Serious adverse events as at least one episode of either the following observed in the ICU: 
- 
Ischemic events defined as either 
o Cerebral ischemia defined as any form of cerebral ischemia on a CT- OR MRI scan 
o Acute myocardial ischemia defined as participant with acute myocardial infarction (ST-
elevation myocardial infarction or non-ST elevation myocardial infarction) or unstable 
angina pectoris according to the criteria in the clinical setting in question (e.g. elevated 
biomarkers, ischemic signs on ECG and clinical presentation) AND the participant 
received treatment as a consequence of this (reperfusion strategies (PCI/thrombolysis) 
OR initiation/increased antithrombotic treatment). 
o Intestinal ischemia defined as ischemia verified by endoscopy OR open surgery OR CT-
angiography. 
o Limb ischemia defined as clinical signs AND need of open/percutaneous vascular 
intervention, amputation OR initiation/increased antithrombotic treatment. 
- 
A new episode of severe acute kidney injury defined as modified KDIGO3 [41]: a 3.0 times 
increase in baseline p-creatinine, or increase in p-creatinine to ≥354 μmol/L, or use of renal 
replacement therapy (any form) in participants who did not receive this before 
randomisation. 
 
-Serious adverse reactions: total number of SARs and number of SARs per participant in the ICU. 
Serious adverse reactions are defined below. 

 
 
 
 
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-Number of days alive without life support at day 90: will be assessed from the use of life support 
including vasopressor/inotrope, mechanical ventilation and renal replacement therapy as defined in 
the inclusion criteria, baseline and daily variables. Total number of days alive without all of the 3 life 
supporting interventions within 90 days after randomisation.  
 
-Days alive and out of hospital at day 90: will be assessed from the discharge date from the index 
hospitalisation, the number of days readmitted to hospital (if any) and date of death, if relevant, 
within the 90-day period 
 
-1-year mortality: landmark mortality 1-year post-randomisation. If the participant has deceased, 
date of death will be registered. 
 
-HRQoL at 1-year (+/- 2 weeks): EQ-5D-5L and EQ-VAS scores (https://euroqol.org/) obtained by 
survey by mail or phone as chosen by the participant. Non-survivors will be given the worst possible 
score. If the participant is incapable of answering the questionnaire (e.g. due to cognitive impairment 
or coma) we will ask proxies to assess HRQoL for the trial participant (proxy point of view) using the 
questionnaire aimed for proxies. Non-survivors will be given the worst possible score. EQ-5D-5L will 
be converted to an index value in combination with the EQ-VAS quantitative measure (0-100 points) 
quantifying self-rated health 
 
-Cognitive function at 1-year (+/- 2 weeks): the Montreal Cognitive assessment (MoCa) MINI score (or 
‘5-miniute protocol’) using the translations of the MoCa full v. 7.1 (http://www.mocatest.org/). The 
score will be obtained in all survivors by interview as this was recommended to be the test of 
cognition in a core outcome set for patients with acute respiratory failure following a modified 
Delphi process involving patients, researchers and clinicians from multiple continents 
(http://www.improvelto.com/). Non-survivors will be given the worst possible score. 
The cognitive function score will be conducted before HRQoL to ensure an equal procedure and 
avoid that patients are disturbed or tired from the HRQoL score. 
If the MoCa MINI, which is validated for phone interview [52], has not been released at the time of 
the first assessment (1-year after randomisation of the first patient), we will adapt the MoCa MINI 
from the MoCa full v. 7.1. 
 
SARs will be defined as follows:  
-General tonic-clonic seizures: stiffening and/or jerking movements of all 4 extremities in a patient 
who becomes or is unconscious in the ICU after randomisation 
 
-Anaphylactic reactions defined as urticarial skin reaction AND at least one of the following observed 
in the ICU after randomisation 
o Worsened circulation (>20% decrease in blood pressure or >20% increase in vasopressor dose) 
o Increased airway resistance (>20% increase in the peak pressure on the ventilation) 
o Clinical stridor or bronchospasm 
o Subsequent treatment with bronchodilators 

 
 
 
 
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-Central pontine myelinolysis seen on CT or MRI scan within the 90-day period after randomisation  
-Hypernatremia defined as p-Na > 159 mmol/L on any plasma sample, including point-of-care testing, 
done in the ICU after randomisation 
 
-Severe hyperchloraemic acidosis defined as pH < 7.15 AND p-chloride > 115 mmol/L on any plasma 
sample, including point-of-care testing, done in the ICU after randomisation 
 
-Severe metabolic alkalosis defined as pH > 7.59 AND SBE > 9 mmol/L on any plasma sample, 
including point-of-care testing, done in the ICU after randomisation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
14.2.1  Pre-screening protocol for Italian sites 
 
For Italian ICUs participating in CLASSIC it will be required to apply the following pre-screening 
criteria for all patients:  
 
- Allergy to study solution or excipient  
- Recent (<3 hours) ventricular fibrillation 
- Sarcoidosis 
- Untreated Addison disease 
- Known hereditary thrombophilic disorders 

 
 
 
 
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- Pregnancy or lactation. Patients with pregnancy or lactation will be excluded. A pregnancy test will 
be done in women in fertile age before enrolment (if not already done during hospital admission) 
 
Patients fulfilling one or more of the above criteria will not be screened for CLASSIC. 
 
 
 
 

 
 
 
 
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14.3 
Appendix 3. Adverse reactions not registered in CLASSIC 
 
The following possible adverse reactions presented in the Danish Summary Product Characteristics 
for the crystalloid solutions will not be registered in the CLASSIC trial as we do consider these to be 
serious conditions: 
 
Normal saline (0.9% NaCl): 
- 
Hypervolemia in itself is not regarded as a SAR, but the potentially serious consequence is 
reflected in the outcome measure days alive without life-support. 
 
Ringer-lactate: 
- 
Sodium retention is not registered as it is not regarded as a SAR. 
- 
Hyperchloraemia is not registered, but is reflected in the SAR hyperchloraemic acidosis. 
 
Ringer-acetate: 
- 
Heart failure is not directly registered, but is reflected in the outcome measure days alive 
without life-support. 
- 
Conjunctivitis is not registered as it is not regarded as a SAR. 
- 
Pulmonary oedema is not directly registered, but is reflected in the outcome measure days 
alive without life-support. 
- 
Rhinitis is not registered as it is not regarded as a SAR. 
- 
Overhydration is not directly registered, but is reflected in the outcome measure days alive 
without life-support. In addition, total fluid balances will be calculated from the daily in- and 
output 
 
PlasmaLyte™: 
- 
Peripheral oedema is not registered as it is not regarded as a SAR 
- 
Pyrexia not registered as it is not regarded as a SAR 
- 
Hypervolemia in itself is not regarded as a SAR, but the potentially serious consequence is 
reflected in the outcome measure days alive without life-support. 
- 
Thrombophlebitis and other reactions at the infusion site are not registered as it is not 
regarded as SARs  
 

 
 
 
 
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14.4 
Appendix 4. Charter for the independent data monitoring and safety 
committee 
 
Introduction 
The DMSC will constitute its own plan of monitoring and meetings. However, this charter will define 
the minimum of obligations and primary responsibilities of the DMSC as perceived of the 
Management Committee (MC), its relationship with other trial components, its membership, and the 
purpose and timing of its meetings. The charter will also outline the procedures for ensuring 
confidentiality and proper communication, the statistical monitoring guidelines to be implemented 
by the DMSC, and an outline of the content of the open and closed reports which will be provided to 
the DMSC. 
 
Primary responsibilities of the DMSC 
The DMSC will be responsible for safeguarding the interests of trial participants, assessing the safety 
and efficacy of the interventions during the trial, and for monitoring the overall conduct of the 
clinical trial. The DMSC will provide recommendations about stopping or continuing the trial to the 
MC of the CLASSIC trial. To contribute to enhancing the integrity of the trial, the DMSC may also 
formulate recommendations relating to the selection/recruitment/retention of participants, their 
management, improving adherence to protocol-specified regimens and retention of participants, and 
the procedures for data management and quality control. 
 
The DMSC will be advisory to the MC. The MC will be responsible for promptly reviewing the DMSC 
recommendations, to decide whether to continue or terminate the trial, and to determine whether 
amendments to the protocol or changes in trial conduct are required. 
 
The DMSC may meet physically or by phone at their own discretion in order to evaluate the planned 
interim analyses of the CLASSIC trial. The interim analyses will be performed by an independent 
statistician selected by the members of the DMSC, NAME (pending) from the Dept. of Biostatistics, 
University of Copenhagen. The DMSC may additionally meet whenever they decide or contact each 
other by telephone or e-mail to discuss the safety for trial participants. The sponsor has the 
responsibility to report the overall number of SARs yearly to the DMSC. The DMSC can, at any time 
during the trial, request the distribution of events, including outcome measures and SARs according 
to intervention groups. Further, the DMSC can request unmasking of the interventions if suggested 
by the data, see section on ‘closed sessions’. The recommendations of the DMSC regarding stopping, 
continuing or changing the design of the trial should be communicated without delay to the MC of 
the CLASSIC trial. As fast as possible, and no later than 48 hours, the MC has the responsibility to 
inform all investigators of the trial and all the sites including patients in the trial, about the 
recommendation of the DMSC and the MC decision hereof.   
 
Members of the DMSC 
The DMSC is an independent multidisciplinary group consisting of clinicians and a biostatistician that, 
collectively, has experience in the management of ICU patients and in the conduct, monitoring and 
analysis of randomized clinical trials. 

 
 
 
 
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DMSC Clinician 
Pending 
 
DMSC Trialist 
Pending 
 
DMSC Biostatistician 
Pending. Dept. of Biostatistics, University of Copenhagen 
 
Conflicts of interest 
DSMC members will fill in and sign a declaration of conflicts of interests (Appendix 10). DMSC 
membership has been restricted to individuals free of conflicts of interest. The source of these 
conflicts may be financial, scientific, or regulatory in nature. Thus, neither trial investigators nor 
individuals employed by the sponsor, nor individuals who might have regulatory responsibilities for 
the trial products, are members of the DMSC. The DMSC members do not own stock in the 
companies having products being evaluated by the CLASSIC trial.  
 
The DMSC members will disclose to fellow members any consulting agreements or financial interests 
they have with the sponsor of the trial, with the contract research organisation (CRO) for the trial (if 
any), or with other sponsors having products that are being evaluated or having products that are 
competitive with those being evaluated in the trial.  
 
The DMSC will be responsible for deciding whether these consulting agreements or financial interests 
materially impact their objectivity. 
 
The DMSC members will be responsible for advising fellow members of any changes in these 
consulting agreements and financial interests that occur during the course of the trial. Any DMSC 
members who develop significant conflicts of interest during the course of the trial should resign 
from the DMSC.  
 
DMSC membership is to be for the duration of the clinical trial. If any members leave the DMSC 
during the course of the trial, the MC will appoint the replacement(s). 
 
Formal interim analysis meetings 
Three formal interim analysis meetings will be held to review data relating to protocol adherence, 
treatment efficacy, participant safety, and quality of trial conduct. The three members of the DMSC 
will meet when day 30 data of 155 participants (10% of sample size) have been obtained, when day 
30 data of 466 participants (30% of sample size) have been obtained and again when 90-day follow-
up data of 777 (50% of sample size) participants have been obtained. 
 
Proper communication 
To enhance the integrity and credibility of the trial, procedures will be implemented to ensure the 
DMSC has sole access to evolving information from the clinical trial regarding comparative results of 

 
 
 
 
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efficacy and safety data, aggregated by treatment group. An exception will be made to permit access 
to an independent statistician who will be responsible for serving as a liaison between the database 
and the DMSC.  
 
At the same time, procedures will be implemented to ensure that proper communication is achieved 
between the DMSC and the trial investigators. To provide a forum for exchange of information 
among various parties who share responsibility for the successful conduct of the trial, a format for 
open sessions and closed sessions will be implemented. The intent of this format is to enable the 
DMSC to preserve confidentiality of the comparative efficacy results while at the same time 
providing opportunities for interaction between the DMSC and others who have valuable insights 
into trial-related issues. 
 
Closed sessions 
Sessions involving only DMSC membership who generates the closed reports (called closed sessions) 
will be held to allow discussion of confidential data from the clinical trial, including information about 
the protocol adherence and the relative efficacy and safety of interventions. To ensure that the 
DMSC will be fully informed in its primary mission of safeguarding the interest of participating 
participants, the DMSC will be blinded in its assessment of safety and efficacy data. However, the 
DMSC can request unblinding from the MC. 
 
Closed reports will include analysis of the volumes of IV fluids (1st and analyses) and the primary 
outcome measure and rates of SAEs and SARs (3rd analysis). These closed reports will be prepared by 
independent biostatistician being a member of the DSMC, with assistance from the trial data 
manager, in a manner that allow them to remain blinded. 
 
The closed reports should provide information that is accurate, with follow-up on mortality that is 
complete to within two months of the date of the DMSC meeting. 
 
Open reports 
For each DMSC meeting, open reports will be provided available to all who attend the DMSC 
meeting. The reports will include data on recruitment and baseline characteristics, and pooled data 
on eligibility violations, completeness of follow-up, and compliance. The independent statistician 
being a member of the DMSC will prepare these open reports in co-operation with the trial data 
manager. 
 
The reports should be provided to DMSC members approximately three days prior to the date of the 
meeting. 
 
Minutes of the DMSC Meetings 
The DMSC will prepare minutes of their meetings. The closed minutes will describe the proceedings 
from all sessions of the DMSC meeting, including the listing of recommendations by the committee. 
Because it is possible that these minutes may contain unblinded information, it is important that they 
are not made available to anyone outside the DMSC.  
 

 
 
 
 
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Recommendations to the Management Committee  
The planned interim analyses will be conducted after participant no. 155 and no. 466 has been 
followed for 30 days and again when no. 777 has been followed for 90 days. 
 
After the interim analysis meetings, the DMSC will make a recommendation to the MC to make 
extraordinary efforts to enforce protocol adherence (1st and 2nd interim analyses) and continue, hold 
or terminate the trial (3rd interim analysis). 
 
The independent DMSC will recommend pausing or stopping the trial if group-difference in the 
primary outcome measure, SARs or SUSARs is found at the interim analyses with statistical 
significance levels adjusted according to the Lan DeMets group sequential monitoring boundaries 
based on O’Brien Fleming alfa-spending function. If the recommendation is to stop the trial the 
DSMC will discuss and recommend on whether the final decision to stop the trial will be made after 
the analysis of all participants included at the time (including participants randomized after 
participant number 777) and whether a moratorium shall take place (setting the trial at hold) in the 
further inclusion of participants during these extra analyses. If further analyses of the participants 
included after 777 participants is recommended the rules for finally recommending stopping of the 
trial should obey the Lan DeMets stopping boundary. 
 
Furthermore, the DMSC can recommend pausing or stopping the trial if continued conduct of the 
trial clearly compromises participant safety. However, stopping for futility to show an intervention 
effect of 15% RRR (or RRI) for mortality will not be an option as intervention effects less than these 
may be clinically relevant as well.    
 
This recommendation will be based primarily on safety and efficacy considerations and will be guided 
by statistical monitoring guidelines defined in this charter and the trial protocol. 
The MC is jointly responsible with the DMSC for safeguarding the interests of participants and for the 
conduct of the trial. Recommendations to amend the protocol or conduct of the trial made by the 
DMSC will be considered and accepted or rejected by the MC. The MC will be responsible for 
deciding whether to continue, hold or stop the trial based on the DMSC recommendations.  
 
The DMSC will be notified of all changes to the trial protocol or conduct. The DMSC concurrence will 
be sought on all substantive recommendations or changes to the protocol or trial conduct prior to 
their implementation. 
 
Statistical monitoring guidelines 
The outcome parameters are defined in the statistical analyses plan in the CLASSIC trial protocol. For 
the two intervention groups, the DMSC will evaluate data on: 
 
First and second interim analyses 
Cumulative volumes of IV fluids given in the ICU excluding IV fluids with medication and nutrition, 
total cumulative volumes of all IV fluids given in the ICU, and rates of protocol violations in the two 
groups. 
Third interim analysis 

 
 
 
 
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Cumulative volumes of IV fluids given in the ICU excluding IV fluids with medication and nutrition, 
total cumulative volumes of all IV fluids given in the ICU, and rates of protocol violations in the two 
groups. 
 
The primary outcome measure 
Mortality in the 90 days after randomisation.  
 
The secondary outcome measures 
- 
The occurrence of SAEs in the ICU 
- 
The occurrence of SARs in the ICU 
The DMSC will be provided with these data from the coordinating centre as: 
- 
Number of participants randomized 
- 
Number of participants randomized per intervention group 
- 
Number of participants stratified per stratification variable per intervention group 
- 
Number of events, according to the outcomes, in the two groups 
 
Based on evaluations of these outcomes, the DMSC will decide if they want further data from the 
coordinating centre and when to perform the next analysis of the data. 
 
For analyses, the data will be provided in one file as described below. 
 
DMSC should yearly be informed about SARs occurring in the two groups of the trial. 
 
The DMSC may also be asked to ensure that procedures are properly implemented to adjust trial 
sample size or duration of follow-up to restore power, if protocol specified event rates are 
inaccurate. If so, the algorithm for doing this should be clearly specified. 
 
 
Conditions for transfer of data from the Coordinating Centre to the DMSC  
The DMSC will be provided with a SAS file containing the data defined as follows: 
 
Row 1 contains the names of the variables (to be defined below). 
 
Row 2 to N (where N-1 is the number of participants having entered the trial) each contains the data 
of one participant. 
 
Column 1 to p (where p is the number of variables to be defined below) each contains in row 1 the 
name of a variable and in the next N rows the values of this variable. 
 
The values of the following variables should be included in the database for the first and second 
interim analyses: 
1. screening_id: a number that uniquely identifies the participant 
2. rand_code: The randomisation code (group 0 or 1). The DMSC is not to be informed on what 
intervention the groups received 

 
 
 
 
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3. Cum_fluid_indic: Cumulative volumes of IV fluids given in the ICU (IV fluids with medication 
and nutrition not included) 
4. Total_Cum_fluid_indic: Cumulative volumes of IV fluids given in the ICU 
5. Protocol_viol_indic: No. of protocol violations in the two groups 
The values of the following variables should be included in the database for the third interim 
analysis: 
1. screening_id: a number that uniquely identifies the participant 
2. rand_code: The randomisation code (group 0 or 1). The DMSC is not to be informed on what 
intervention the groups received 
3. Cum_fluid_indic: Cumulative volumes of IV fluids given in the ICU (IV fluids with medication 
and nutrition not included) 
4. Total_Cum_fluid_indic: Cumulative volumes of IV fluids given in the ICU 
5. Protocol_viol_indic: No. of protocol violations in the two groups 
6. day_90_indic: 90 day-mortality indicator (2 = censored, 1=dead, 0=alive at day 90) 
7. SAE_indic: SAE indicator (1 = one or more SAEs, 0 = no SAE) 
8. SAR_indic: SAR indicator (1 = one or more SARs, 0 = no SAR) 
 
 

 
 
 
 
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14.5 
Appendix 5. Informed consent, Denmark 
In Denmark temporarily incompetent patients will be enrolled after informed consent from one 
medical doctor, who is independent of the trial (first trial guardian). As soon as possible after 
enrolment, consent will be obtained from the patient’s next of kin and a second medical doctor 
(second trial guardian). The second trial guardian must be different from the first trial guardian and 
also independent of the trial. Participants, who regain consciousness, will be asked for informed 
consent as soon as possible. The process leading to the achievement of informed consent will be in 
compliance with all applicable regulations. The consenting party will be provided with written and 
oral information about the trial so he/she is able to make an informed decision about participation in 
the trial. The information will be given in a separate room, and the consenting party has the right to 
bring a companion.  
 
Written information and the consent form will be subjected to review and approval by the relevant 
ethic committees.  
 
Lack of informed consent from the participant’s next of kin 
If information about the participant’s next of kin is not available after inclusion, the investigator will 
seek information from e.g. the participant’s general practitioner, the police, nursing homes etc. In 
these situations it may take 1-2 weeks to conclude that no next of kin can be identified. If no one is 
identified and the participant remains incompetent the trial intervention will be discontinued. All 
initiatives to identify the participant’s next of kin will be documented in patient files, logs or similar.  
 
Lack of informed consent from the participant’s next of kin and the participant deceases  
If the participant deceases before informed consent has been obtained (due to rapid progression of 
critical illness or because the participant’s next of kin is not yet identified) and the participant has 
been correctly included in the trial, the collected data will be kept for analysis.   
 
Deviation from the standard informed consent 
According to the standard informed consent form from the National Ethics Committee regarding 
competent participants, the participant can choose not to receive information about the data 
collected during the trial. However, the purpose of this trial is not to generate new knowledge about 
the specific participant, so we find that this question is redundant, and have omitted the question 
from the consent form to spare the participant from making unnecessary decisions.  
Trial personnel  
 
 
 
 
         
Screening will be performed by medical doctors or medical students working under the responsibility 
of a trained medical doctor.  
Collection of informed consent will be performed by all trial personnel (study nurses, medical 
students, doctors). If questions arise during informed consent, responsible study personnel can be 
reached through a 24-h hotline. All personnel with functions in the CLASSIC trial will be trained and 
approved according to GCP-guidelines before engaging in the trial. 
 

 
 
 
 
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14.5.1            Informed consent, Sweden 
 
Informed consent 
 
 
 
 
                   
The patients in septic shock will receive written and oral information of the study and they will be 
asked to give oral consent before enrolment. As/if they recover they will receive written and oral 
information about the study and they will give written informed consent to continue in the study. 
 
Reporting of Serious Adverse Events 
 
 
                                              
Serious adverse events are any adverse events that results in death, are life-threatening, require 
hospitalization or prolongation of existing hospitalization, results in persistent or significant disability 
or incapacity, or is a congenital anomaly or birth defect. We will specifically be registering ischemic 
events (cerebral, cardiac, intestinal or limb ischemia and new episodes of severe acute kidney injury. 
SAEs will be reported to the sponsor within 24 hours, according to LVFS 2011:19. Any SAE that is not 
listed in the RSI will be considered as unexpected, including all fatal events, and be reported as 
Suspected Unexpected Serious Adverse Reactions (SUSARs). They will be reported by the sponsor to 
the Medical Product Agency within 7 days after the report reached the sponsor. 
 
Monitoring 
 
 
 
 
 
                    
The monitoring and data verification plan is developed by Copenhagen University Hospital GCP Unit, 
Denmark. National investigators will ensure local monitoring in adherence to the monitoring plan 
and national regulations. The local investigators will ensure that the monitors and the regulatory 
personnel will have access to patient records/ source data for monitoring and inspections. 
 
 
 
 
 
  
 
 
 

 
 
 
 
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14.6 
Appendix 6. Simplified Mortality Score for the Intensive Care Unit 
(SMS-ICU) 
In trial settings, the variables are measured in the 24-h period before randomisation; further details 
are presented in Appendix 2 and in [43].  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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14.7 
Appendix 7. Co-enrolment 
 
Based upon an updated critical appraisal of the literature, the CLASSIC Management Committee 
endorses and encourages co-enrolment in the CLASSIC trial. The following issues have been 
considered. 
 
Ethical considerations 
Preventing eligible patients from co-enrolment in trials, which they would authentically value 
participating in, and whose material risks and benefits they understand, violates their autonomy - 
and thus contravenes a fundamental principle of research ethics [53].  
 
Permitting co-enrolment is in accordance with existing recommendations for the conduct of 
trustworthy clinical practice guidelines, taking into account benefits and harms, quality of evidence, 
values and preferences (of patients or their proxies) and cost considerations, as outlined by the 
Institute of Medicine, the Guideline International Network, and according to the GRADE 
methodology [54, 55, 56]. 
 
Patient relatives have limited concerns about co-enrolment [57]. 
 
General considerations 
Critically ill patients receive many different interventions in addition to the trial intervention because 
of acute and chronic illness. Consequently, the potential for interactions is a prerequisite in clinical 
trials in critically ill patients, and co-enrolment is thus little different from what occurs in single-
enrolment trials [53]. 
 
In large pragmatic trials, like the CLASSIC trial, other interventions will be given at random and are 
therefore difficult to control for. If interaction in fact is an issue, it may be better controlled for if 
patients are co-enrolled and randomised to more than one intervention.  
 
Factorial design trials allow detailed assessment of interactions between interventions, and are 
considered cost-efficient, as two or more treatments are assessed for the price of one [58]. Co-
enrolment trials and factorial design trials share many similarities [53].  
 
A pre-planned sub-study will assess the impact of co-enrolment in the CLASSIC trial, and thus 
generate valuable knowledge on the topic of co-enrolment.  
 
Clinical research with a potential to inform and improve clinical practice is valuable and should be 
supported. More high-quality clinical research can be conducted in a timely fashion and more 
information can be generated to guide clinical practice, if co-enrolment is permitted [59]. 
 
Scientific and statistical considerations 
Pragmatic clinical trials allowing inclusion of a broad range of trial participants and options for drug 
treatments and other therapies (co-enrolment) have higher external validity/generalizability than 
non-pragmatic trials with restrictions regarding trial participants and co-enrolment [60].  
 
Non-pragmatic trials with restrictions regarding study participants and co-enrolment are exposed to 
drugs and other treatments in a less clinically relevant setting where interactions are largely 
uncontrolled and poorly evaluated. Co-enrolment in pragmatic trials facilitates evaluation of clinically 
relevant and patient-important interactions [53].  
 

 
 
 
 
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Co-enrolment into two or more trials does not invalidate the original randomization of the individual 
trials. Separate analysis of each individual trial, ignoring the issue of co-enrolment into the other trial, 
will retain the balance of patient characteristics expected by standard random assignment within 
each trial [53]. 
 
The National Institute of Health supports co-enrolment [60]; so does the Canadian Critical Care Trials 
group (http://www.ccctg.ca/Home.aspx) and the Australian New Zealand Intensive Care Society’s 
Clinical Trial Group (http://www.anzics.com.au/Pages/CTG/CTG-home.aspx). We have co-enrolment 
agreements with the two latter research groups. 
 
Co-enrolment into two or more trials does not seem to affect the natural course of the disease of the 
other condition being studied [53]. 
 
Co-enrolment does not appear to influence patient safety or trial results [61, 62]. 
 
Empirically, co-enrolment has a small effect on study power [53].   
 
In conclusion, we highly support and encourage co-enrolment because of overall benefit, including 
ethical, practical and scientific benefit, and no evidence of harm. 
 
 

 
 
 
 
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14.7.1 
Co-enrolment agreement form  
 
In general we will encourage engagement in research projects other than the CLASSIC trial. 
Please, fill in the information of the trial to be evaluated as counterpart for co-enrolment with 
CLASSIC, and send it by e-mail to contact@cric.nu. 
 
Once we have received the information below, we will contact the principal/coordinating 
investigator of the trial and facilitate exchange of protocols and other relevant documents between 
the Management Committees. You will find a list of titles already considered for co-enrolment by 
clicking http://www.cric.nu/co-enrolment-list/ 
 
We have prepared the form for only one trial, but please feel free to copy as many forms as you 
need.  
 
 
a. Official full/short title of the project:  
 
 
 
 
 
b. Contact information of principal/coordinating investigator of the trial: 
 
Name:  
 
E-mail: 
 
 
 
 
 

 
 
 
 
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14.8 
Appendix 8. List of proposed sub-studies 
 
• The effect of co-enrolment on the intervention effect in the CLASSIC trial 
• The effect of restrictive fluid management on glycocalyx degradation - a preplanned substudy of 
the CLASSIC trial 
• Temporal changes in plasma lactate concentration in critically ill patients with septic shock 
treated with restrictive compared to standard fluid therapy – a substudy to the CLASSIC trial  
 
14.8.1 
Approved substudy protocols   
a. The effect of restrictive fluid management on glycocalyx degradation - a preplanned 
substudy of the CLASSIC trial  
 
b. Temporal changes in plasma lactate concentration in critically ill patients with septic 
shock treated with restrictive compared to standard fluid therapy – a substudy to the 
CLASSIC trial 

 
 
 
 
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14.8.1. a. The effect of restrictive fluid management on glycocalyx 
degradation - a preplanned substudy of the CLASSIC trial 
 
Jens Christensen1, Michelle Chew2, Johan Mårtensson1, Suvi Vaara3, Tom Eirik Mollnes4, Søren Erik 
Pischke4, Peter Hjortrup5, Theis Lange6, Maria Cronhjort1 
 
1 Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Sweden 
2 Department of Biomedical and Clinical Sciences 
3 HUS Perioperative, Intensive Care and Pain Medicine, Department of Diagnostics and Therapeutics 
Anestesiologian yksikkö, University of Helsinki, Helsinki University Hospital Area 
4 Department of Immunology, University of Oslo 
5 Department of Intensive Care, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. 
6  Section of Biostatistics, University of Copenhagen, Copenhagen, Denmark 
 
 
Background 
The endothelial glycocalyx is a carbohydrate rich protein layer on the inside of the vessels. It is 
attached to the vessel wall via proteoglycans like syndecan and glypican. It consists of 
glycosaminoglycans (hyaluronic acid (HA), heparan sulfate (HS) and chondroitin sulfate) and soluble 
plasma components. HS is the most common glycosaminoglycan and might have great structural 
heterogeneity as it can differ in length and sulfation pattern . HS influences cellular signaling through 
the fibroblast growth factor pathway (3).  The glycocalyx has several important functions, as it 
influences the interaction between blood cells and the vascular wall. As the glycocalyx Is degraded, 
e.g. during inflammation, soluble fragments of HS are thought to influence endothelial cell signaling 
and the systemic response to critical illness (2). HA is elevated in critical illness and especially in 
sepsis. It is also a marker of fibrotic liver disease, both due to increased deposition and reduced 
clearance, as it is cleared from the blood in the liver sinusoids (4).  
 

 
 
 
 
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Fig. 1. Structure of the endothelial glycocalyx/endothelial surface layer (ESL).  
(a) Endothelial glycocalyx thickness is larger than the endothelial cell 
itself, as demonstrated by electron microscopy of ruthenium-red labeled rat myocardial capillaries. In vivo, the 
glycocalyx forms an even more substantial ESL, with thickness >1 mm. Figure 1a used with permission from van 
den Berg et al. (5)  
(b) Pathological degradation of the glycocalyx/ 
ESL during critical illnesses (such as sepsis) causes not only local endothelial injury, but also releases biologically 
active heparan sulfate fragments 
into the circulation that may influence signaling processes in an endocrine fashion. For simplicity, chondroitin 
sulfate and hyaluronic acid are not 
shown. a4 and b4 refer to glycosidic bonds connecting constituent saccharides. Inset: structure of a heparan 
sulfate octasaccharide fragment, 
demonstrating potential sites of sulfation within constituent disaccharide units. 
Figure 1b used with permission from SAGE publishing open access (2). 
 
It is possible that the endothelial dysfunction observed in septic patients is due to glycocalyx 
dysfunction. Moreover, inflammatory cytokines increase the secretion of angiopoietin-2 (Ang-2) from 
the endothelial cells (6), and Ang-2 has been shown to increase glycocalyx shedding (7). Ang-2 levels 
are associated with mortality in sepsis (8). 
Degradation of the endothelial glycocalyx can either be proved directly by electron microscopy or 
indirectly by elevated levels of its degradation products; syndecan-1, heparan sulfate and hyaluronic 
acid. It has been shown to be degraded in different clinical situations, like exposure for endotoxins 
(9), infection (10), critical illness (11) and septic shock (12). It is also damaged by intravenous fluid 
bolus therapy(13) (14). In a septic mice model, the thickness of the glycocalyx significantly diminished 
during induced sepsis (15). High levels of Syndecan-1 has been suggested as a marker to identify 

 
 
 
 
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patients at higher risk of negative effects of fluid therapy (11). In a study of patients in septic shock in 
the emergency department there was an association between fluid balance the first three hours and 
the levels of HA, but it is uncertain to what extent fluid therapy contributes to degradation of the 
glycocalyx. (10). 
The aim of this study is to study the effect of restrictive fluid management compared to standard 
care on the levels of hyaluronan. We will also analyze the association between high hyaluronan, 
heparan sulfate, syndecan-1 TNFR, IL-6and Ang-2 levels at arrival to the ICU with fluid overload. Thus, 
we will explore the possibility of using degradation products of the glycocalyx as markers of patients 
with high risk to suffer from complications from fluid overload.  
 
Method 
This will be a sub-study in an RCT of restrictive fluid management in patients with septic shock, the 
CLASSIC trial, which is a continuation of the CLASSIC pilot trial(16).  In the CLASSIC trial1554 patients 
will be randomized to restrictive or standard fluid therapy during the ICU stay.  
Blood samples will be drawn at T0 (during the first hour after enrolment), T1 (the first morning after 
inclusion) and T2 (the second morning) and T3 (at ICU discharge (within 24 hours before discharge)). 
We will collect blood samples for blood for RNA analyses, EDTA-plasma, Citrate-plasma and serum in 
four test tubes. Blood samples will be handled in accordance to the attached handling procedure 
(attachment 1). All samples will be kept in a research freezer at -80C until further analysis at one 
central laboratory. We aim to analyze markers of glycocalyx break-down products: heparan sulfate, 
syndecan-1, hyaluronic acid, IL-6, TNFR, Ang-2.  
 
Outcomes 
Study 1. The primary outcome is the difference in concentration of hyaluronan at the first morning 
after enrolment(T1) in the restrictive group compared with the control group, adjusted for the T0 
values.  
Secondary outcomes are associations between glycocalyx degradation product levels (heparan 
sulfate, syndecan-1, hyaluronic acid, IL-6, TNFR, Ang-2.) at arrival in the ICU and fluid balance after 24 
and 72 hours in the ICU. 
 
Statistical methods and sample size calculation 
Baseline clinical and demographic data will be analyzed using chi-square or Fisher’s exact test for 
categorical data, and the Wilcoxon rank-sum test for continuous data. The baseline and first follow-
up measure of the concentration of hyaluronan will be modeled using a mixed effect linear model 

 
 
 
 
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with a person specific random effect and an interaction between time of follow-up measure (interval 
since first measurement) and treatment group, the stratification variables for the randomization: 
hematological or metastatic cancer (Y/N) and trial site as fixed effects. Where biomarkers are 
normally, or log-normally distributed, mixed model analyses will be employed to analyze the pattern 
over time. Differences in the pattern of expression of each biomarker between the groups will be 
tested by the interaction of time and group in the model. Additional covariates will be added to 
adjust for treating center, illness severity (SAPS 3), cumulative fluid balance and comorbidities 
(Charlson comorbidity index).  
 
A conservative sample size calculation reveals that with an assumed standard deviation of 29 ng/ml  
of hyaluronan concentration (established in previous comparable studies (10), a sample size of 200 
would yield 80% to detect a group difference of 11.5 ng/l at T1. True power will be higher as the 
planned analysis also controls for baseline values, but as the exact size of this correlation is not 
known we restrict our self to the conservative sample size calculation. To account for drop-out or 
similar we will add a further 10% and therefore target a sample size of 220 equally distributed 
between the two groups.  
We will use the Jakobsen-Lange adjustment to adjust for several secondary outcomes (17) where αi 
for each outcome is αi=0.05/((n+1)/2) which is an adjustment halfway between no adjustment and 
full Bonferroni adjustment with n being the number of co-primary outcomes and secondary 
outcomes respectively. 
 
Importance 
This study might elucidate the mechanism through which fluid therapy affects the glycocalyx. 
Glycocalyx markers might also be able to identify patients at risk for fluid leakage and thus more 
complications from fluid therapy. This might be a way to personalize fluid therapy in septic patients.  
 
References 
1. Schott U, Solomon C, Fries D, Bentzer P. The endothelial glycocalyx and its disruption, protection and 
regeneration: a narrative review. Scandinavian journal of trauma, resuscitation and emergency medicine. 
2016;24:48. 
2. Oshima K, Haeger SM, Hippensteel JA, Herson PS, Schmidt EP. More than a biomarker: the systemic 
consequences of heparan sulfate fragments released during endothelial surface layer degradation (2017 Grover 
Conference Series). Pulmonary circulation. 2018;8(1):2045893217745786. 

 
 
 
 
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3. Esko JD, Selleck SB. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annual review of 
biochemistry. 2002;71:435-71. 
4. Yagmur E, Koch A, Haumann M, Kramann R, Trautwein C, Tacke F. Hyaluronan serum concentrations are 
elevated in critically ill patients and associated with disease severity. Clinical biochemistry. 2012;45(1-2):82-7. 
5. van den Berg BM, Vink H, Spaan JA. The endothelial glycocalyx protects against myocardial edema. 
Circulation research. 2003;92(6):592-4. 
6. Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S, Thurston G, et al. Angiopoietin-2 sensitizes 
endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nature medicine. 
2006;12(2):235-9. 
7. Lukasz A, Hillgruber C, Oberleithner H, Kusche-Vihrog K, Pavenstadt H, Rovas A, et al. Endothelial glycocalyx 
breakdown is mediated by angiopoietin-2. Cardiovascular research. 2017;113(6):671-80. 
8. Siner JM, Bhandari V, Engle KM, Elias JA, Siegel MD. Elevated serum angiopoietin 2 levels are associated with 
increased mortality in sepsis. Shock. 2009;31(4):348-53. 
9. Okada H, Takemura G, Suzuki K, Oda K, Takada C, Hotta Y, et al. Three-dimensional ultrastructure of capillary 
endothelial glycocalyx under normal and experimental endotoxemic conditions. Crit Care. 2017;21(1):261. 
10. Smart L, Macdonald SPJ, Burrows S, Bosio E, Arendts G, Fatovich DM. Endothelial glycocalyx biomarkers 
increase in patients with infection during Emergency Department treatment. J Crit Care. 2017;42:304-9. 
11. Puskarich MA, Cornelius DC, Tharp J, Nandi U, Jones AE. Plasma syndecan-1 levels identify a cohort of 
patients with severe sepsis at high risk for intubation after large-volume intravenous fluid resuscitation. J Crit 
Care. 2016;36:125-9. 
12. Nelson A, Berkestedt I, Schmidtchen A, Ljunggren L, Bodelsson M. Increased levels of glycosaminoglycans 
during septic shock: relation to mortality and the antibacterial actions of plasma. Shock. 2008;30(6):623-7. 
13. Chappell D, Bruegger D, Potzel J, Jacob M, Brettner F, Vogeser M, et al. Hypervolemia increases release of 
atrial natriuretic peptide and shedding of the endothelial glycocalyx. Crit Care. 2014;18(5):538. 
14. Powell MF, Mathru M, Brandon A, Patel R, Frolich MA. Assessment of endothelial glycocalyx disruption in 
term parturients receiving a fluid bolus before spinal anesthesia: a prospective observational study. 
International journal of obstetric anesthesia. 2014;23(4):330-4. 
15. Kataoka H, Ushiyama A, Akimoto Y, Matsubara S, Kawakami H, Iijima T. Structural Behavior of the 
Endothelial Glycocalyx Is Associated With Pathophysiologic Status in Septic Mice: An Integrated Approach to 
Analyzing the Behavior and Function of the Glycocalyx Using Both Electron and Fluorescence Intravital 
Microscopy. Anesth Analg. 2017;125(3):874-83. 
16. Hjortrup PB, Haase N, Bundgaard H, Thomsen SL, Winding R, Pettila V, et al. Restricting volumes of 
resuscitation fluid in adults with septic shock after initial management: the CLASSIC randomised, parallel-group, 
multicentre feasibility trial. Intensive Care Med. 2016;42(11):1695-705. 
17. Jakobsen JC, Wetterslev J, Winkel P, Lange T, Gluud C. Thresholds for statistical and clinical significance in 
systematic reviews with meta-analytic methods. BMC medical research methodology. 2014;14:120. 

 
 
 
 
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14.8.1. b. Temporal changes in plasma lactate concentration in critically ill 
patients with septic shock treated with restrictive compared to 
standard fluid therapy – a substudy to the CLASSIC trial 
 
Christian Ahlstedt1, Praleene Sivapalan2, Miroslaw Kriz3, Morten Hylander Møller2, Marek Nalos3,  
Olav Rooijackers1, Maria Cronhjort4, Anders Perner2, Jonathan Grip1 
 
1 Perioperative Medicine & Intensive Care, Karolinska University Hospital Huddinge, Sweden 
2 Department of Intensive Care, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. 
3 Department of Anesthesia and Intensive care Medicine, Charles University Hospital Pilsen, Czech Republic 
4 Department of Clinical Science and Education, Södersjukhuset, Karolinska Insititutet, Stockholm, Sweden 
 
Background and study rationale 
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to 
infection (1) and septic shock is a subgroup of sepsis with particularly severe circulatory and 
metabolic abnormalities. 
Elevated plasma lactate is part of Sepsis-3 definition of septic shock. Its correlation with worsened 
outcome in sepsis and septic shock is well known. The current surviving sepsis campaign guidelines 
recommend immediate and serial lactate measurements until normalization (2). 
A systematic review from 2016 by Vincent JL et al., which included 34 observational sepsis studies, 
showed that decrease in lactate concentration over time was consistently associated with lower 
mortality rates in all subgroups of patients (3). Hyperlactatemia is in clinical practice often thought to 
indicate that the human body is unable to uphold aerobic metabolism, due to tissue hypoxia and/or a 
deranged micro-circulation. This is often taken as an indication that a sufficient amount of 
resuscitation fluid has not yet been given to the patient. However, there are many other causes for 
hyperlactatemia, such as increased glycolysis, decreased clearance, and adrenergic stress response 
(4, 5). 
In a Dutch multicenter randomized controlled trial from 2010 lactate-guided therapy in 
hyperlactatemia (albeit from various causes) reduced mortality, but interestingly the lactate levels 
did not differ significantly between the groups (6). 
The current paradigm states that treatment with fluids decreases elevated concentrations of plasma 
lactate. Therefore, it is somewhat surprising that the post-hoc analysis of the Conservative versus 
Liberal Approach to fluid therapy of Septic Shock in Intensive Care (CLASSIC) feasibility trial (7) 

 
 
 
 
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showed no significant differences in plasma lactate levels between the two treatment groups even 
though there was >1 L difference in received fluids between the groups. The trial itself was not 
powered to investigate this outcome, but the finding is worth investigating in the main study. 
 
Trial hypotheses and outcomes 
The objective of this sub-study of the CLASSIC-trial is to use the randomization to either restrictive or 
standard fluid therapy to study temporal changes of lactate and time to normalization in patients 
with septic shock. 
 
Research questions: 
1. Does the lactate values differ between the two intervention groups in terms of 
a. Temporal changes in concentrations over time (based on all available plasma lactate analyses 
during the first 72 hours). 
b. Differences in delta-values (ΔLactate/h) over time (Calculated according to the method described 
under method & design). 
c. Differences in time to normalization (<2 mM/L) or discharge/death. 
2. Same question(s) as above but comparing the cohort of patients with a higher baseline lactate (>4 
mmol/L) in the two treatment arms. 
 
Hypotheses: 
1. Restrictive fluid therapy will neither significantly affect the delta-lactate nor time to normalization 
in patients treated for septic shock compared with standard (more liberal) fluid therapy 
2. Patients in septic shock with a higher baseline lactate >4mmol/L will not decrease lactate levels 
faster with standard fluid therapy compared with restrictive fluid therapy. This is a sub-group analysis 
of 1). 
3. A high baseline lactate and/or a low ΔLactate/h is associated with a higher mortality irrespective of 
fluid treatment. 
 
Outcome measure(s) 
1. Primary outcomes are the differences in plasma lactate concentrations and ΔLactate/h during the 
first 72 hours between the two treatment arms in the CLASSIC trial. 
2. Secondary outcomes are the time to normalization of lactate, length of intensive care unit (ICU) 
stay and 90-day mortality in restrictive vs standard group of the CLASSIC trial. 
 

 
 
 
 
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Method and design 
All retrospective serial time-stamped point-of-care blood gas analyses from patients included in the 
CLASSIC trial will be collected and analyzed for the first 72 hours or until death or discharge from the 
ICU (depending on the availability from the centers). 
Baseline characteristics (age, sex, body weight, pre-existing conditions and lab), daily fluid balance, 
urine output, treatment (mechanical ventilation, renal replacement therapy, vasopressors) and 90-
day mortality data of included patients will be available from the CLASSIC trial database. 
Normally distributed continuous data will be presented as mean +/- standard deviation and 
independent Student´s t test will be used to compare means. Mann-Whitney U test will be used to 
compare mean ranks between groups if data shows non-normality assessed by the Shapiro-Wilk test. 
Such data will be presented as median (interquartile range). Data of categorical variables will be 
analyzed with Chi-square or Fisher’s exact test as appropriate. A P-value of <0,05 will be considered 
as a statistically significant difference between groups for the primary outcomes. 
ΔLactate/h is defined as (baseline lactate/baseline lactate – subsequent lactate) / baseline lactate x 
(100 / time interval(h)). Baseline lactate value is the lactate concentration at the time of 
randomization (from the electronic case report form). We will compare the groups using a mixed 
model for repeated measures to deal with missing values. Logistic multiple regression analysis for 
baseline lactate,ΔLactate/h and mortality adjusted for allocation treatment group and important risk 
factors (age, co-morbidities, severity of disease et cetera). 
Centers will provide data in pseudonymized form. Data will be stored in an encrypted hard drive. Key 
for decryption and identification will be stored in a fire safe locked cabinet. 
 
Sample size estimate 
The Swedish centers have to date included 130 patients. Outside of Sweden the two hospital regions 
of Zealand, Denmark have included 604 patients and the University Hospital of Pilsen, Czech 
Republic, have included 50 patients. The estimated sample size will all together consist of 784 
patients. The number of blood gas analyses will vary depending on patient and including center.  
 
References: 
1. Singer M et al (2016) The Third Inter- national Consensus definitions for sepsis and septic shock (Sepsis-3). 
JAMA 315(8):801–810 
2. Evans L et al, Surviving sepsis campaign: international guidelines for management of sepsis and septic shock 
2021, Intensive Care Medicine (2021) 47:1181–1247 

 
 
 
 
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3. Vincent JL et al, The value of blood lactate kinetics in critically ill patients: a systematic review. Crit Care 
2016:20;257 
4. Hernandez G et al, The ten pitfalls of lactate clearance in sepsis, Intensive Care Medicine (2019) 45: 82-85 
5. Garcia-Alvarez et al, Sepsis-associated hyperlactatemia, Critical Care 2014, 18:503 
6. Jansen TC et al, LACTATE study group. Early lactate guide-therapy in intensive care unit patients: a 
multicenter, open-label, randomized controlled trial. Am J Respir Crit Care Med 2010;182:752-761. 
7. Hjortrup et al, Effects of Fluid Restriction on measures of circulatory efficacy in adults with septic shock. Acta 
Anaesthesiol Scand 2017 Apr;61(4):390-398 
 
 

 
 
 
 
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14.9 
 Appendix 9. Preliminary results of our systematic review 
 
Preliminary results of our systematic review on restrictive vs. standard of care/more liberal fluid 
resuscitation in patients with septic shock. 
 
Pending 
 
 

 
 
 
 
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14.10 
Appendix 10. Trial flow chart 
Please refer to the Consort Statement for more information (http://www.consort-statement.org/). 
The flowchart should be modified to reflect the flow of participants in the trial. The flowchart (n= ) 
will be filled in at the end of the trial. 
 
 
 
Analysed (n= ) 
• Excluded from analysis (give reasons) (n= ) 
• Lost to follow-up (give reasons) (n= ) 
• Discontinued intervention (give reasons) (n= ) 
Allocated to intervention (n= ) 
• Received allocated intervention (n= ) 
• Did not receive allocated intervention (give 
reasons) (n= ) 
• Lost to follow-up (give reasons) (n= ) 
• Discontinued intervention (give reasons) (n= ) 
Allocated to intervention (n= ) 
• Received allocated intervention (n= ) 
• Did not receive allocated intervention (give 
reasons) (n= ) 
Analysed (n= ) 
• Excluded from analysis (give reasons) (n= ) 
Allocation 
Analysis 
Follow-Up 
Excluded (n= ) 
• Meeting specified exclusion criteria (n= ) 
• Other reasons (n= ) 
Randomised (n= ) 
Assessed for eligibility (n= ) 
Enrolment 

 
 
 
 
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14.11 
Appendix 11. International Committee of Medical Journal Editors 
(ICMJE) form for potential conflict of interest 
 

 
 
 
 
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14.12 
Appendix 12. LOG of protocol changes 
 
Protocol version 
  
List of changes 
Version 2.0, March 12th 
2018 
Danish Medicines 
Approval 
March 20th 2018 
 
Version 2.1, May 3rd 
2018 
Approved 
Protocol update after Danish Ethics 
Committee approval: p19 and p20 
”Ethical justification and rationale”: 
detailed description of the population 
and first trial guardian.  
Version 2.2, Nov 13th, 
2018 
Approved 
- 
Change of 3 site investigators  
- 
Specification in 3.4 ‘participant 
timeline’ 
- 
Addition of Swedish consent 
procedures section 9.1.2 
- 
Updated organization diagram 
appendix 14.1 
- 
Specification of  collection of 
consent appendix 14.5 
(addition that consent can be 
collected by medical students 
trained in the protocol) 
- 
Addition of Swedish consent 
procedures appendix 14.5 
Version 2.3, June 19th 
2019 
 
- 
Change of trial manager name 
- 
Addition of 1 Danish site 
investigator  
- 
Addition of national investigator 
for Brussels 
- 
Specification of ‘End of trial’ in 
new paragraph 3.5  
- 
Specification in 4.3 that 
withdrawal by proxy/participant 
will follow national regulations 
and those described in 4.3.1 
- 
Specification in 8.4 on 
reporting of serious adverse 
reactions/events. 
- 
Updated Steering Committee 
diagram 
- 
Updated definition secondary 
outcome ‘intestinal ischemia’ 
- 
Language specification in the 
definitions of inclusion and 
exclusion criteria in 14.2 
- 
Language specification in one 
baseline variable in 14.2 
- 
Specification of acute kidney 
injury component of the 
composite secondary outcome 
‘SAE’, HRQoL and MoCA 
secondary outcomes in 14.2  

 
 
 
 
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- 
Specification of extracted fluid 
data for the interim analyses in 
14.4 
Version 2.4, May 23th, 
2022 
 
- 
Specification in appendix 8: 
updated list of CLASSIC 
substudies including substudy 
protocols