DetectIon of Severe Sepsis In PATients With Neurological haemorrhagE (The DISSIPATE Study)
Conditions: Sepsis, Hemorrhage Brain
Sponsor: National University Hospital, Singapore

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 1 of 12 
 
 
 
 
 
STUDY PROTOCOL 
 
PROTOCOL TITLE: 
DetectIon of Severe Sepsis In PATients with neurological haemorrhage (The DISSIPATE 
Study) 
 
PROTOCOL NUMBER:  
1 
 
PROTOCOL VERSION:  
Version 2 
PROTOCOL DATE:  
14 May 2020 
 
PRINCIPAL INVESTIGATOR:  
Dr Loh Ne-Hooi Will, Senior Consultant, Department of Anaesthesia, National University 
Hospital 
CO-INVESTIGATORS 
Dr Christina Yip Yew Ching, Post-Doctorate Fellow, Department of Laboratory Medicine, 
National University Hospital 
Dr Eng Soo Yap, Consultant, Department of Haematology-Oncology, National University 
Hospital 
Dr Shen Jiayi, Medical Officer, Department of Anaesthesia, National University Hospital 
 
 
 
 
STUDY SITE: 
National University Hospital 
 
 
 
 
 
 
 
O F F I C I AL  U S E  O N L Y  
Doc Name : Protocol Template 
Doc Number : 207-002 
Doc Version : 
03 
Date : 01 June  
09 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 2 of 12 
 
Table of Contents 
 
1. Rationale and Background…………………………………………………………………………………………………….3 
       2.    Study Aims…………………………………………………………………………………………………………………………….4 
       3.    Study Design ............................................................................................................................... 5 
       4.    Patient Recruitment ................................................................................................................... 6 
       5.    Data Collection ........................................................................................................................... 9 
       6.    Study Outcomes ......................................................................................................................... 8 
       7.    Data Analysis…………………………………………………………………………………………………………………………8 
       8. Sample Size and Statistical Methods……………………………………………………………………………………..8 
       9.    Ethical Considerations…………………………………………………………………………………………………………..9 
     10.    Retention of Study Documents……………………………………………………………………………………………..9 
 
 
 
 

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1. Rationale and Background 
Rationale  
Systemic inflammatory response syndrome (SIRS) and pyrexia are frequent conditions after brain 
injuries making conventional detection of sepsis extremely inaccurate1,2. Early detection of sepsis 
helps in reducing the secondary injury to brain and the same time accuracy in detection of sepsis is 
needed to avoid indiscriminate antimicrobial usage and further antimicrobial resistance. Current 
commonly used biological markers of sepsis like white cell count (WBC) and CRP (C-Reactive Protein) 
is unspecific for sepsis in this population as they are usually elevated due to the surgery or trauma. 
Other biological markers such as procalcitonin (PCT), tumour necrosis factor (TNF) and Interleukin-6 
have been evaluated but the costs can be prohibitive, delayed due to the processing time and non-
specific2. Pelinka et al has also showed that traditional sepsis markers like CRP and PCT are inaccurate 
in head injuries3,4. Microbiological culture of bacteria is currently the gold standard for sepsis but 
processing time takes at least 24 hours and is not specific and is frequently at risk of contamination if 
not sampled correctly. The indiscriminate use of broad-spectrum antibiotics promotes the selection 
of antibiotic resistance and has further complications. At the same time, patients presenting with a 
Sequential Organ Failure Assessment (SOFA) score of 2 or more (which is the new definition of sepsis) 
have an overall mortality risk of 10% with presumed infection which is greater than overall mortality 
of 8.1% for ST- segment elevation myocardial infarction5. So, clinicians have daily difficulties in 
deciding if a patient is septic when faced with critically ill patients especially with brain injuries and 
SIRS. 
 
Background  
Activated Partial Thromboplastin Time (APTT) clot waveform analysis (CWA) and WBC activation 
analysis by flow cytometry-based method are new and very low cost biological tests proposed for the 
diagnosis of sepsis in the general ICU population. Recent advances in coagulation analysers have 
enabled the ability to obtain optical profiles of the clotting process for routine tests such as 
Prothrombin Time and APTT within 1 hour of the sample reaching the laboratory. The analysis using 
real time optical transmittance to provide more information from the clotting assay than just an 
endpoint clotting time. The profiles plot change in light transmittance against time, producing a ‘wave-
like’ plot. Abnormal CWA have been seen in many conditions such as trauma and Disseminated 
intravascular coagulation (DIC) and especially in sepsis. Studies have shown that they predict for 
severe sepsis, septic shock and DIC 6,7. Pilot studies in the general ICU have shown promise in the 
detection of severe sepsis8. Toh et al also found that this biphasic waveform was due to the rapid 
precipitation of CRP, and very-low-density lipoprotein (VLDL) in the presence of calcium ions. CRP 
belongs to a family of proteins known as pentraxins, and its level rapidly increases under conditions 
of infection or tissue damage9. Presence of abnormal waveforms has been confirmed in severe sepsis 
in studies performed in the general ICU9 and in personal observations in University Hospital Aintree. 
It was identified that biphasic waveform was more accurate than PCT and CRP for differentiating 
patients with severe sepsis and septic shock, with 90% sensitivity and 92% negative predictive value6,7. 
These biphasic waveform values were significantly more abnormal during day 1–3 in septic 
nonsurvivors than in survivors and nonseptic nonsurvivors and exhibited the best specificity (91%) and 
negative predictive value (98%) for the prognosis of sepsis-related mortality on day 36. There are no 
studies have examined the diagnostic value of these novel sepsis measures in the neurosurgical 
population of subarachnoid hemorrhage, traumatic brain injury and other intracranial hemorrhages. 
Routine analysis of WBC using flow cytometery-based method has been shown to be low cost, rapid 
and can be incorporated as part of the routine full blood count (FBC) analysis. During stress or 
infection, less mature neutrophil forms enter circulation, including an increased number of bands. This 
is referred to as a left shift, which is defined as an elevated immature/total granulocyte ratio or an 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 4 of 12 
 
elevated neutrophil band count. Neutrophil and monocyte activation has been shown to correlate 
well in laboratory studies and in pilot clinical studies on septic ICU patients10. Linssen et al showed that 
using the Sysmex XE-2100 analyser allowed the real time study of monocyte and neutrophil activation 
by using the routine fluorescence-flow cytometer of the analyser. The information about cell shape, 
cell membrane content and cell granularity, thus giving an idea as to WBC activation status. The 
‘Neutrophil X’ (NEUT-X) parameter measures neutrophil granularity. The more the cells degranulate 
(in response to sepsis), the lower the parameter value becomes. The ‘Neutrophil Y’ (NEUT-Y) 
parameter measures the production and release of proteins (reactive oxygen intermediates and 
cytokines) from neutrophils by a change in nucleic acid content. The immature granulocyte parameter 
(IG) parameter and neutrophil reactive index (NEUT-RI) also correlates with sepsis.  Wiemann et al 
(2015) has used a composite score, Intensive care Infection Score (ICIS) in medical-surgical ICU 
patients to differentiate between with infection and without infection with high sensitivity and 
specificity11. The ICIS comprised of five blood-cell derived parameters that characterize the early 
innate immune response: (I) mean fluorescence intensity of mature (segmented) neutrophils; (sNGI) 
(II) difference in haemoglobin concentration between newly formed and mature red blood 
cells;(dCHC) or delta-Hemoglobin (III) total segmented neutrophil count;(sN#) (IV) antibody secreting 
lymphocyte count;(ASL) (V) immature granulocyte count(aIG). In the study, an ICIS score of more than 
3 was considered as positive score for sepsis using a modified fully automated routine fluorescence 
hematology analyser (XN-10; Sysmex, Kobe, Japan). This can be measured during the WBC 
measurement and does not require any additional blood sampling.  
 
2. Study Aims and Significance 
 
Primary Aim 
To validate the use of APTT CWA and ICIS, as early sepsis markers for neurosurgical ICU patients 
suffering subarachnoid haemorrhage, traumatic brain injury and other intracranial haemorrhages.   
Secondary Aim 
To examine the evolution of CWA, immuno-parameters (KL-6, SP-A, MIG, presepsin) and various WBC 
activation markers over the time in relation to diagnosis of sepsis, development of positive blood 
cultures and mortality or recovery. Blood parameter measurements using a 3-part and 5-part 
differential analyser will be performed.  
 
Potential Risks 
This will purely be an observational study of changes in laboratory parameters and the  
research participants do not deviate from the current standard of care. However minimal  
risks are anticipated for this study as all additional blood samples collected are from arterial lines 
inserted as part of routine care for patients in the SICU.  
 
Potential Benefits 
There is no assurance that the research participants will benefit from participation in this study. 
However, their participation in this study may add to the medical knowledge by exploring novel early 
predictors and validation of laboratory parameters in the management of sepsis. This will greatly 
complement current clinical monitoring of patients in the SICU without subjecting the patients to 
additional invasive tests. 

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Study Significance 
The research study is to explore novel early predictors and validation of laboratory parameters in the 
management of sepsis. This will greatly complement current clinical monitoring of patients in the SICU 
without subjecting the patients to additional invasive tests.  
 
3. Study Design 
 
This is a prospective observational study. 150 subjects with the admission diagnosis of neurological 
haemorrhage (e.g. subarachnoid haemorrhage, intracerebral haemorrhage etc), admitted to SICU of 
National University Hospital, Singapore, who are expected to stay for more than 48 hours, will be 
recruited and enrolled. The proposed workflow for this study is highlighted in Figure 1. No adverse 
events are expected as a result of this study as patients do not deviate from the current standard of 
care. Frequency of blood sampling will be stipulated at day 1/2/3/4/5 to draw clinical relevance. An 
additional 0.5 tablespoonful (7.7ml) of blood will be taken daily from each subject as well as residual 
blood from routine laboratory test blood samples. In total, an additional volume of 2.5 tablespoonful 
(38.5ml) of blood will be collected from each subject over a period of 5 days. Residual blood refers to 
any leftover blood samples collected for routine laboratory tests which will be shared and used to 
carry out additional blood tests as shown in Table 1. All ICU patients will have arterial lines inserted 
as part of routine care, so there will not be pain from the blood sampling. In the current study, 
measurements will be made on the routine laboratory haematology, coagulation and immunoassay 
analyzers. Research blood parameters that are not usually reported as indicated in the background 
section will be used for the correlation and association analysis to sepsis. Over a period of 2 years, 
these blood samples will be collected from 150 patients, stored in -80 degrees’ freezer and then 
used for batch standard laboratory tests. The remaining blood samples will be stored in NUH in -80 
degrees’ freezer over a period of 5 years for future biomedical research use.  
In addition, the study team will also be collecting clinical and demographic data of recruited subjects 
from medical records for purposes of this study. 
 
 
 
 
 
 
 
 
 
 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 6 of 12 
 
 
 
Patient admitted to Surgical ICU for neurological haemorrhage 
 
Screened for eligibility (refers to using the subject eligibility checklist for preliminary screening to 
identify potential subjects by reviewing databases or medical records) 
 
Enrolment of patient after obtaining informed consent  
 
Standard Laboratory Test orders (FBC, CRP, Creatinine and Blood cultures as indicated)  
 
Daily clinical Assessments and Treatment monitoring# 
 
Mortality / Discharge from ICU  
 
Figure 1. Proposed flow chart for research project  
#The diagnosis of sepsis is obtained from the treating physician or the commencement of antibiotics 
and confirmed by microbiological cultures (routinely performed during this period as the standard of 
care to detect sepsis). The treating physician will not be involved in data analysis of CWA, immuno-
parameters and WBC infection score. Results will be stratified based on patients who have no SIRS, 
SIRS but no microbiological evidence of sepsis (via blood, sputum, urine cultures etc), sepsis (SIRS + 
microbiological evidence) and retrospectively analysed. The patient will be treated according to the 
treating physician’s routine standard of care practices in the best interest of the patient. No potential 
adverse events are expected as this will purely be an observational study of changes in laboratory 
parameters.  
Source of sepsis secondary to ventilator associated pneumonia is defined according to the new CDC 
guideline for definition which includes 
 
Definition of ventilator associated event plus, on or after calendar day 3 of mechanical 
ventilation and within 2 calendar days before or after the onset of worsening oxygenation, 
following criteria to be met 
 
Criteria one: Positive culture of one of the specimens meeting quantitative or semi 
quantitative thresholds (Endotracheal aspirate >105CFU/ml; Bronchoalveolar lavage > 
104CFU/ml; Lung tissue >104 CFU/ml and protected specimen brush >103 CFU/ml without need 
for purulent secretions 
 
Criteria two: Purulent respiratory secretions plus organism identified from one of the above 
specimens without sufficient growth to meet criterion 1 
 
Definition of bacteraemia is by presence of positive blood cultures            
 
Catheter associated Urinary tract infection is defined as presence of positive urinary 
microscopic examination of more than 5 WBC from the freshly placed urinary catheter long 
with positive urinary cultures 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 7 of 12 
 
 
 
Table 1. Blood volume required for investigation 
# Actual blood investigation may vary depends on patient’s clinical conditions 
Volume marked with *, **, $ indicate sharing of blood tube for testing. 
Note:  
- 
7.7ml of additional blood (highlighted in yellow) is taken for this study daily over 5 days for 
these additional blood tests: APTT, Procalctitonin and Serological/Inflammatory markers (KL-
6, SPA, MIG, Presepsis). All other bloods are routinely taken for routine investigations during 
their SICU stay. 
- 
Out of the 7.7ml of additional blood, 5ml of blood drawn will be shared and used for these 
blood tests: Procalcitonin and Serological/Inflammatory markers (KL-6, SPA, MIG, Presepsis) 
as marked with this symbol $.  
 
 
 
 
 
 
Blood volume collection and testing: 
#Standard of Care Blood 
Investigations  
DAY 1 
DAY 2 
DAY 3 
DAY4 
DAY 5 
FBC (EDTA/Lavendar top) 
3ml* 
3ml* 
3ml* 
3ml* 
3ml* 
CRP (SSTII/Red Top) 
5ml** 
- 
5ml** 
- 
5ml** 
Blood cultures (routine 
ICU care for fever) 
10ml 
- 
10ml 
- 
10ml 
CREATININE (SSTII/Red 
Top) 
5ml** 
5ml** 
5ml** 
5ml** 
5ml** 
Additional blood 
investigations: 
 
 
 
 
 
FBC (3-Part Diff) (EDTA 
/Lavendar Top) 
3ml* 
3ml* 
3ml* 
3ml* 
3ml* 
PTINR/APTT (Citrate/Blue 
Top) 
2.7ml 
2.7ml 
2.7ml 
2.7ml 
2.7ml 
PROCALCITONIN 
(SSTII/Red Top) 
5ml$ 
- 
5ml$ 
- 
5ml$ 
Serological/Inflammatory 
Markers (KL-6, SPA, MIG, 
Presepsis)(SSTII/Red Top) 
 
5ml$ 
 
5ml$ 
 
5ml$ 
 
5ml$ 
 
5ml$ 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 8 of 12 
 
 
4. Patient Recruitment 
 
List the number of subjects to be enrolled 
A total target number of 150 subjects with the admission diagnosis of neurological haemorrhage 
(e.g. subarachnoid haemorrhage, intracerebral haemorrhage etc), admitted to SICU of National 
University Hospital, Singapore, who are expected to stay for more than 48 hours, will be recruited 
and enrolled. 
 
Criteria for recruitment 
The SICU anaesthetist on duty would make first contact with potential participants of the research 
study. After obtaining consent, the SICU anaesthetic team will then make the necessary referral to 
the study team members who will then screen for their eligibility based on the subject eligibility 
checklist. These potential participants will be recruited by face-to-face contact via their legally 
acceptable representative when they are in SICU. 
 
Inclusion criteria for subjects: 
 
Adults 21 years and above 
 
Clinical/radiological suspicion or confirmation of neurological haemorrhage 
 
Exclusion criteria for subjects: 
 
Age below 21 years 
 
Prisoners 
 
Known pregnancy 
 
Do-not-attempt resuscitation status 
 
Requirement for immediate surgery 
 
Active chemotherapy/neutropenia (Neutrophil count <1.0 x 109/L) 
 
Immuno-compromised  
 
Haematological malignancy 
 
Treating physician deems aggressive care unsuitable 
 
Unable to provide informed consent or comply with study requirements 
 
 
Patient withdrawal 
Patients will be allowed to withdraw anytime during the study without comprising their care. 
 
Subject Replacement 
Subjects who drop out will not be replaced to reach the minimum target number. 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 9 of 12 
 
 
5. Data Collection 
 
Data will be collected for all tests performed (raw data format and instrument files) and the clinical 
data includes 
 
Past medical history and demographic data 
 
Presence of SIRS; timing of sepsis and septic shock 
 
APACHE II, SOFA and other disease severity scoring systems 
 
Routine blood tests such as CRP and/or PCT 
 
Timing of Blood cultures and therapies (Inotropes and antibiotics etc.) 
 
Longitudinal test results to include PT/aPTT CWA, FBC, WBC infection scores including ICIS and 
related parameters (marked as ICU day 1 to day 5), immuno-parameters (KL-6, SP-A, MIG, 
Presepsin) 
 
Complications 
 
Length of ICU stay and mortality 
 
6. Study Outcomes 
 
The primary analysis of the study is to examine a number of laboratory test parameters and its 
association to sepsis as the clinical outcome. The associations between the presence or absence of 
the biphasic wave form in CWA, WBC activation parameters and clinical relevance of immuno-
parameters at baseline and subsequent diagnosis of sepsis.  
Secondary analysis will examine the potential predictive value of evolution of CWA and WBC 
parameters over time in relation to the diagnosis of sepsis, development of positive blood cultures 
and mortality. 
 
7. Data Analysis 
Data Quality Assurance 
The study team will ensure that the data obtained from this research is accurate, complete and 
reliable.  
Data entry and Storage 
The data will be entered into excel sheet, and then be stored and handled. 
 
 
 
 

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 10 of 12 
 
8. Sample size and Statistical methods 
 
Determination of sample size 
This is a pilot study to determine the resources needed for a full scale study, thus 150 has been 
selected as the recruitment target. 
 
Statistical and Analytical Plans 
In order to assess the ability of baseline CWA, immuno-parameters and ICIS to discriminate between 
septic and non-septic patients, a receiver operator characteristic (ROC) curve will be constructed for 
each measure against presence of sepsis as determined by the treating physician (SIRS), and by 
microbiological culture of bacteria (severe sepsis). As a result, the sample size is based on that needed 
to evaluate the area under the ROC curve. We assume that an AUC of 0.7 (while better than chance) 
would be insufficiently discriminating, but as one aim of the study is to estimate the AUC, a value of 
0.85 has been set as the value expected. If this is the true value, then the sample size to rule out a 
value of 0.7 or less, with 80% power and one-sided significance of 5% is 98 patients, assuming the 
proportion of patients with sepsis is at least 25%.  
Simple descriptive statistics (proportions and accompanying confidence intervals) will be used to 
assess complication rates and other frequency data. Continuous measures will be assessed using mean 
and standard deviation (or median and interquartile range if the distribution is non-symmetrical). A 
number of further exploratory analyses may then be undertaken. Simple line plots of CWA and WBC 
activation parameters for each patient over time and summary measures or relevant repeated 
measures regression techniques may be considered. If feasible this may include the analysis of the 
relationship between sepsis confirmation and measures of CWA, immuno-parameters and ICIS over 
time. Time to event analyses may also be undertaken using standard techniques (e.g. Kaplan-Meier 
plots, Cox proportional hazards models) to compare the survival prospects and/or time to sepsis for 
patients with and without SIRS or severe sepsis at baseline.  
 
 
9. Ethical Considerations 
 
Informed consent 
Informed consent will be obtained prior to performing any study-related procedures. The informed 
consent process will be taken at SICU via the potential participants’ legally acceptable representative 
(LAR). The LAR would be given sufficient time to decide whether to let them participate in the 
research study. The consent will be conducted in a private room in SICU together with participants’ 
LAR. The wishes of the participant’s LAR must also be respected if they chose not to allow the 
participant to take part in the research study. 
 
IRB review 
Each participating institution must provide for the review and approval of this protocol and the 
associated informed consent documents by the IRB/ NHG DSRB/ SGH CIRB.  

Study Protocol Number 1, Version 2, Dated 14/5/2020                                                Page 11 of 12 
 
Confidentiality of Data and Patient Records 
For hardcopy data, they will be stored in designated locked cabinet(s) or room(s) that are accessible 
to authorized study personnel only. 
For electronic data, they will be stored on in a secured computer that is password protected. The 
databases will not contain subject identifiers and the data linking subject identifiers and the subject 
identification codes will be stored separately. 
 
10. 
Retention of study documents 
Records for all participants, including CRFs, all source documentation (containing evidence to study 
eligibility, history and physical findings, laboratory data, results of consultations, etc.) as well as IRB 
records and other regulatory documentation should be retained by the PI in a secure storage facility 
for 6 years or minimum duration of retention period as specified by the institutional policy. The 
records should be accessible for inspection and copying by authorized authorities. 
 
 

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