The MicroRESUS Study: An Observational Study to Examine the Effects of Circulatory Shock and Resuscitation on Microcirculatory Function and Mitochondrial Respiration After Cardiovascular Surgery.
Conditions: Shock, Cardiovascular Diseases, Critical Illness
Sponsor: University of Pennsylvania

Protocol date: 6/1/2020 
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Title: The MicroRESUS Study: An Observational Study to Examine the Effects of Circulatory Shock and 
Resuscitation on Microcirculatory Function and Mitochondrial Respiration After Cardiovascular Surgery.  
 
 
NCT05330676    
Unique Protocol ID: 843614 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator: 
 
John Greenwood, MD 
Assistant Professor, Department of Emergency Medicine 
Department of Anesthesiology & Critical Care 
Center for Resuscitation science 
Perelman School of Medicine at the University of Pennsylvania 
Philadelphia, PA 19104-4399 
Email: john.greenwood@pennmedicine.upenn.edu 
 

Protocol date: 6/1/2020 
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Abstract 
Background 
Despite current resuscitation strategies, circulatory shock and organ injury after cardiac surgery occur in 25-40% 
of patients. Goal-directed resuscitation after cardiac surgery has generated significant interest, but clinical 
practice to normalize hemodynamic variables including mean arterial pressure, cardiac filling pressures, and 
cardiac output may not reverse microcirculation abnormalities and do not address cellular dysoxia. Recent 
advances in technology have made it possible to measure critical components of oxygen delivery and oxygen 
utilization systems in live human tissues and blood cells. The MicroRESUS study will be the first study to measure 
microcirculatory and mitochondrial function in patients with circulatory shock and link these findings with clinical 
outcomes.  
 
Methods and analysis 
This will be a prospective, observational study that includes patients undergoing elective cardiovascular surgery 
with cardiopulmonary bypass (CPB). Microcirculation will be quantified with sublingual incident dark field 
videomicroscopy. Mitochondrial respiration will be measured by performing a substrate–uncoupler–inhibitor 
titration protocol with high resolution respirometry on peripheral blood mononuclear cells at baseline and serial 
timepoints during resuscitation and at recovery as a possible liquid biomarker. Plasma samples will be preserved 
for future analysis to examine endothelial injury and other mechanisms of microcirculatory dysfunction. Thirty-
day ventilator and vasopressor-free days (VVFDs) will be measured as a primary outcome, along with sequential 
organ failure assessment scores, and other clinical parameters to determine if changes in microcirculation and 
mitochondrial respiration are more strongly associated with clinical outcomes compared to traditional 
resuscitation targets.  
 
This will be the first prospective study to examine both microcirculatory and mitochondrial function in human 
patients with circulatory shock undergoing cardiac bypass and address a key mechanistic knowledge gap in the 
cardiovascular literature. The results of this study will direct future research efforts and therapeutic development 
for patients with shock. 
 

Protocol date: 6/1/2020 
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Introduction 
Despite current resuscitation practices, circulatory shock and perioperative organ injury after cardiac surgery 
occur in 25-40% of patients [1,2]. Traditionally, causes of shock after cardiovascular surgery with 
cardiopulmonary bypass are classified by macrocirculatory derangements (e.g. low cardiac output, vasoplegia, 
hypovolemia) [3]. Interventions to normalize hemodynamic variables, including mean arterial pressure, cardiac 
filling pressures, and cardiac output restore the large vessel (macrocirculatory) pressure and flow targets but 
may not reverse underrecognized disruptions in microcirculatory blood flow and oxygen utilization. Current 
methods to estimate the balance of oxygen delivery (DO2) relative to demand (VO2) include blood gas-derived 
calculations and the measurement of downstream biomarkers of anaerobic metabolism such as blood lactate. 
Using these methods, previous literature has concluded that lactic acidosis after cardiac surgery is unlikely 
related to inadequate oxygen delivery [4]. Unfortunately these inferences fail to consider the presence of regional 
blood flow derangements caused by pathologic microcirculatory heterogeneity, which are also associated with 
severity of postoperative lactic acidosis and organ injury [5,6]. To resolve this important clinical discrepancy, a 
deeper understanding of the determinants of oxygen transport pathways and oxygen utilization during health, 
shock, and resuscitation are needed. 
 
The microcirculation is composed of a network of vessels including arterioles, capillaries, and venules <100 μm 
in diameter where red blood cells (RBCs), leukocytes, and plasma components interface with the vascular 
endothelium to allow metabolic substrate exchange. Changes in microcirculatory blood flow can be caused by 
inflammatory-mediated vascular endothelial injury, microthrombosis, or an inadequate balance between 
vasoconstrictive and vasodilating agents leading to a global or heterogeneous reduction in capillary blood flow 
[7,8].  Incident dark field (IDF) videomicroscopy is a novel, handheld method that can directly image the human 
microcirculation in real time. Current generation IDF videomicroscopy has improved imaging resolution 
compared to previous generation devices, and can detect up to 30% more capillaries compared to side stream 
imaging [9,10]. As a result, important determinants of tissue oxygenation, such as microcirculatory diffusive and 
convective properties, can now be more accurately quantified. 
 
Blood gas derived calculations of VO2/DO2 balance may provide false clinical reassurance as they cannot identify 
microvascular injury, microvascular shunting, and other mechanisms of dysoxia which may contribute to cell 
injury and organ dysfunction in patients with shock [11]. Advancements in high resolution respirometry now make 
it possible to quantify mitochondrial respiration rapidly and reliably in live tissues [12]. Nucleated blood cells 
(platelets and peripheral blood mononuclear cells) are readily accessible and can be used as surrogates to study 
cellular respiration in acute care illnesses such as acute heart failure, hemorrhagic shock, sepsis, and patients 
with ischemic reperfusion injury [13–15]. It is unclear if deficiencies in oxygen delivery and utilization occur 
independently or concomitantly in patients with shock. Studies that simultaneously examine mitochondrial 
respiration and microcirculatory function, which are also tied to clinical outcomes, are vitally important to guide 
future research efforts in therapeutic development and perform effective interventional trials. 
 
Materials and methods 
Study objective 
The primary objective of the MicroRESUS study is to evaluate the microcirculatory and mitochondrial function in 
patients following elective cardiovascular surgery with cardiopulmonary bypass, to determine if these parameters 
outperform traditional biomarkers and global hemodynamic measurements to predict clinical outcomes. 
 
Study design and setting 
This is a prospective, observational, single center study with repeated measures from baseline through 30-days 
after surgery at the University of Pennsylvania in Philadelphia, PA, USA.  
 
Patient screening 
Patients will be screened 24-48 hours prior to surgery using the published operative schedule. First-case elective 
coronary artery bypass graft (CABG) or valvular surgeries will be considered for study enrollment, pending 
availability of research personnel to complete the study protocol. Efforts will be made to diversify subject 
enrollment to accurately reflect the general cardiovascular patient population.  
 

Protocol date: 6/1/2020 
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Informed consent 
Patients will be approached for consent by telephone one day prior to surgery or in person while in the 
preoperative area on the day of surgery. Consent will be obtained by the Principal Investigator or trained research 
personnel. Patient signatures will be obtained digitally via the REDcap data management system [16]. A digital 
copy of the consent form will be sent to the patient in addition to being retained by the study team. 
 
Sample size and power 
Using data from our previous foundational work as well as unpublished pilot data, we anticipate a 1:1 allocation 
of patients to the high (PVD > 22mm/mm2) and low group (PVD < 22 mm/mm2). We will need a sample size of 
at least 134 subjects to detect at least a 2-day difference in ventilator and vasopressor-free days (VVFDs) with 
a β=0.8, using a one-sided t-test α=0.05 [5,6]. We will enroll a total of 140 subjects to allow for a 5% loss to 
follow-up (surgical delay, ICU delay, cancelled surgery, etc.) and exclusion due inadequate microcirculation 
video quality (S1 Appendix). 
 
Inclusion criteria 
Adult patients (>18 years old) receiving elective CABG or valvular surgery requiring cardiopulmonary bypass are 
eligible for enrollment. Post-operative patients with circulatory shock will be identified by having: 
1. Either vasopressor-dependent hypotension or low cardiac output requiring inotropic support 
2. Signs of end-organ injury or impaired tissue perfusion defined by one of the following criteria: 
a. Normothermic patients with a capillary refill time > 3 seconds 
b. Serum lactate > 2 mmol/dL 
c. Mixed venous oxygen saturation (SvO2) < 60% 
 
Exclusion criteria 
Patients will be excluded if they are unable to tolerate sublingual microcirculatory flow imaging (e.g., non-
intubated patients dependent upon oxygen by facemask, poor mouth opening), receiving an emergent 
procedure, have an active malignancy, or mitochondrial disorder.  
 
Timeline and techniques for data collection 
Data for each subject will be collected at time points outlined in Error! Reference source not found.. Prior to 
surgery, baseline biologic samples, microcirculation imaging, and clinical data will be obtained. Repeated 
measurements will be obtained in the ICU and surgical floor. Long-term clinical outcomes will be recorded at 30-
days or upon discharge. 
Fig 1. Timeline of data collection. 
 
Demographic and resuscitation data 
Demographic values including age, gender, and ethnicity will be recorded. Preoperative risk scores including the 
STS (Society of Thoracic Surgeons) mortality score and euroSCORE II (European System for Cardiac Operative 
Risk Evaluation) will be calculated [51,52]. Intraoperative data including cardiopulmonary bypass time, cross 
clamp time, blood product use, intravenous fluid administration, and vasoactive administration will be recorded. 
ICU clinical data (hemodynamics, laboratory testing, etc.), resuscitation data (intravenous fluids, vasoactive 
administration, blood transfusion, etc.), and clinical outcomes (VVFDs, ICU length of stay, hospital LOS, etc.) 
will be recorded as indicated in Error! Reference source not found.. Sequential organ failure assessment 
(SOFA) scores will be calculated prior to surgery (baseline), as well as 24 and 72 hours after surgery.  

Protocol date: 6/1/2020 
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Figure 1 
 
Physiologic and pharmacologic data 
Systemic hemodynamic data, perfusion data, and blood gas measurements will be collected upon ICU 
admission, then hourly during the first 6 hours of postoperative care. Cardiac output (CO), cardiac index (CI), 
central venous pressure (CVP), pulmonary artery pressure (PAP), and mixed venous oxygen saturation (SvO2) 
will be monitored continuously using a pulmonary artery catheter (Edwards Lifesciences LLC, Irvine, CA, USA). 
Arterial blood pressure will be measured using a standard invasive arterial line. Intraoperative and post-operative 
administration of blood products, intravenous fluids, vasoactive agents (vasopressor and inotropes), and 
sedatives will be recorded at each time point. 
  
Incident Dark Field Microscopy 
Sublingual microcirculation imaging will be performed using handheld incident dark field (IDF) videomicroscopy 
(CytoCam, Braedius Medical BV, the Netherlands) at four time points during the enrollment period. Imaging will 
be performed by the Principal Investigator or trained research personnel. Video sequences are obtained by 
placing the CytoCam device should sublingual space and maneuvered so that pressure and motion do not result 
in image artifact (Error! Reference source not found.). A series of successive video clips (3-5 clips of at least 
120 frames or 6 seconds in length) should be captured in distinct areas of the sublingual space to account for 
vessel heterogeneity. Focus and lighting during video capture may be adjusted to optimize image acquisition. 
Baseline (T0h) imaging will be obtained in the preoperative area prior to surgery on the day of the scheduled 
operation. Repeated measurements will be obtained upon arrival to the ICU (0-2 hours after surgery, T2h), during 
ongoing resuscitation (2-4 hours post-op, T4h), and on post-operative day one after recovery (T24h).  
 

Protocol date: 6/1/2020 
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Fig 2. A. Experimental setup with patient in supine position during IDF measurement. B. Anatomical 
sublingual triangle where measurements are obtained. 
 
Analysis of IDF videomicroscopy 
Prior to analysis, video quality will be assessed using the 6-factor Massey quality score, which uses a 
semiquantitative assessment of each video for appropriate illumination, duration, focus, content, stability, and 
pressure. Only videos with Massey scores of <10 will be included for further analysis [17]. Three videos with the 
best quality score will be selected for further processing. All IDF images will be coded then analyzed using an 
offline, dedicated software (Automated Vascular Analysis v3.02, Microvision Medical, The Netherlands). 
Microvascular flow index (MFI), microcirculatory heterogeneity index (MHI), total vessel density (TVD), proportion 
of perfused vessels (PPV), and perfused vessel density (PVD). Individual microvessel flow will be scored as 
0=no flow, 1=intermittent flow, 2=sluggish flow, or 3=continuous flow. Vessels will be considered perfused if they 
are scored as either sluggish or continuous flow. To ensure only vessels contributing to tissue gas exchange 
and metabolism are included, only vessels < 20 µm in diameter will be analyzed. This process follows the current 
standard for microcirculation measurement and analysis [18]. Interobserver variation will be tested periodically 
in at least 10% of the subject videos to ensure minimal scoring heterogeneity between investigators.  
 
Biological samples  
Blood gas samples will be drawn into a commercial, pre-heparinized 1 mL blood sampler then immediately 
analyzed by an ABL90 FLEX automatic blood gas analyzer (Radiometer America Inc., Brea, California, USA). 
Arterial blood gas samples will be obtained by clinical staff every 1-2 hours for the first 6 hours after ICU 
admission based on current clinical practice. Central venous blood gas measurements will also be obtained by 
the study team at the time of microcirculation measurement. Hemoglobin, hematocrit, pH, PaO2, PaCO2, and 
glucose are also reported on each blood gas analysis. After 6 hours, blood gas analysis will be performed as 
needed by the clinical team. For biomarker and mitochondrial function analysis, enrolled patients will undergo 
phlebotomy with volumes of 15 mL drawn into K2EDTA tubes. Blood samples will then be centrifuged at room 
temperature using Ficoll-PaqueTM PLUS (GE) and Leucosep tubes (Greiner Bio-one). Plasma specimens will be 
stored at -80ºC for later evaluation of the mechanisms of microcirculatory dysfunction. Peripheral blood 
mononuclear cells (PBMCs) will undergo further analysis and processing as detailed below.  
 
Mitochondrial respiration 
A population of PBMCs will be obtained and analyzed from the plasma buffy coat within 1 hour of blood draw. A 
cell count and viability will be calculated using the Cell Countess II (Invitrogen) with trypan blue exclusion. 
Between 4-5 x 106 PBMCs will be used for respiration analysis and residual PBMCs will be processed and stored 

Protocol date: 6/1/2020 
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at-80º C for future quantitative PCR. Unless otherwise specified, all reagents will be obtained from Sigma-Aldrich 
and Invitrogen. 
 
Mitochondrial respiration will be analyzed using an Oroboros O2k-FluoRespirometer (Oroboros Instruments, 
Innsbruck, Austria) with a substrate–uncoupler–inhibitor titration (SUIT) protocol and  MiR05 buffer [19]. The 
SUIT protocol measures oxidative phosphorylation capacity with electron flow through complex I (CI) and 
complex II (CI + CII) using malate, pyruvate, glutamate, and flavin adenine dinucleotide-linked substrate 
succinate in the presence of adenosine diphosphate. The addition of digitonin allows for the measurement of 
specific complex-linked activity. Oligomycin, an inhibitor of the ATP synthase, uncouples respiration from ATP-
synthase activity to measure respiration where the O2 consumption is dependent on the leakiness the 
mitochondrial membrane and back-flux of protons into the mitochondrial matrix independent of ATP synthase 
(LEAKCI+CII). Maximal convergent non-phosphorylating respiration of ETSCI+CII is evaluated by titrating the 
protonophore, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. ETSCI+CII is considered a stress test for 
mitochondria, a marker of mitochondrial respiratory reserve. Non-phosphorylating respiration specifically through 
CII (ETSCII) is achieved through the addition of rotenone, an inhibitor of CI. The complex III (CIII) inhibitor 
antimycin-A is added to measure the residual non-mitochondrial oxygen consumption, and this value is 
subtracted from each of the measured respiratory states to provide only mitochondrial respiration. Complex IV 
(CIV)-linked respiration will be measured by the addition of ascorbate with N,N,N,N-tetramethyl-
phenylenediamine. The CIV inhibitor sodium azide will be added to reveal the chemical background that is 
subtracted from the N,N,N,N-tetramethyl-phenylenediamine-induced oxygen consumption rate. Mitochondrial 
reactive oxygen species production will be measured using the Amplex UltraRed method [20]. All data will be 
acquired using DatLab 7 (Oroboros Instruments, Innsbruck, Austria) and respiration value will be normalized to 
cell count. 
 
Outcome measures 
Our hypothesis is that patients with poor post-operative sublingual functional capillary density (defined as a PVD 
< 22 mm/mm2 and MHI > 0.4) will have a higher degree of postoperative cardiovascular and pulmonary organ 
injury compared to patients with normal postoperative microcirculation. The primary outcome for this study will 
be VVFDs during the first 30 days after surgery. This outcome was chosen because our elective cardiac surgery 
patients are managed using early recovery after surgery (ERAS) protocols, with a goal to  be extubated and 
weaned off vasopressors within 24 hours after surgery [21,22]. VVFDs will be calculated as a reverse count of 
consecutive days without requiring ICU-level respiratory or vasopressor support (Error! Reference source not 
found.). Our research group has used a similar primary outcome in a previous clinical trial [23]. The day of 
operation (day zero) will not be included as all patients undergoing cardiovascular surgery with CPB require 
mechanical ventilation and vasoactive medications on the day of surgery. Ventilator days will include mechanical 
ventilation via endotracheal tube or tracheostomy, high-flow nasal cannula > 40 liters/minute with an FiO2 > 
40%, or non-invasive positive pressure ventilation not prescribed at home. Non-invasive positive pressure 
ventilation and HFNC will be included as these both require the patient to remain in our ICU. Vasoactives include 
epinephrine, norepinephrine, vasopressin, phenylephrine, dobutamine, or milrinone at any dose. Subjects in 
need of respiratory or vasopressor support on day 30, or die before day 30 will be assigned zero VVFDs. If the 
patient is discharged prior to hospital day 30 a “last status carried forward” approach will be used. Secondary 
outcomes will include sequential organ failure assessment (SOFA) score on day 1, day 3, diagnosis of acute 
kidney injury (defined as an increse in serum creatinine > 0.3 mg/dL within 2 days), and hospital length of stay.  

Protocol date: 6/1/2020 
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Fig 3. Examples of VVFD scenarios to determine clinical outcomes. 
 
Exploratory outcomes will examine the relationship of microcirculatory function with common clinical biomarkers 
such as lactate, SvO2, venous-to-arterial PCO2 gap, capillary refill time, and lactate to pyruvate ratio. Markers of 
endothelial injury and inflammation such as soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble 
intercellular adhesion molecule 1 (sICAM-1), e-selectin, IL-8, IL-10, and other inflammatory cytokines will be 
measured. We will also examine mitochondrial complex function in human blood cells, comparing individual 
complex (I, II, III, and IV) function and reactive oxygen species generation between patients with and without 
post-operative shock.  
 
Statistical and analysis plans 
Continuous variables characterizing demographical data, microcirculation data, and mitochondrial respiration 
measurements, and outcomes data will be reported as means with standard deviations if normally distributed or 
medians with interquartile ranges if not normally distributed. Categorical variables will be represented as 
frequencies and proportions. To examine the predictive performance of selected variables for the primary 
outcome, we will construct receiver operator characteristic curves for threshold values of PVD, MHI, lactate, 
SvO2, mean arterial pressure, and cardiac index. A Youden index will be calculated to determine the best cutoff 
value for determining prolonged VVFDs.  
 
Linear regression modeling will be used to examine the relationship between L/P ratio and postoperative 
microcirculation variables. We will perform univariate analyses on candidate predictor variables of L/P ratio 
including PVD, MHI, LFTs, creatinine, CPB time, cross clamp time, and catecholamine administration. Multiple 
linear regression analysis will be used to model the effect of significant predictors. Repeated measure ANOVA 
will be used to compare changes in microcirculatory variables, mitochondrial respiration, and mitochondrial 
reactive oxygen species production over time. To adjust for multiple comparisons, post-hoc pairwise Tukey 
Kramer t-tests will be performed. All analyses will use statistical software (SAS version 15.1, Cary, NC; Prism v 
9.0, Graph-Pad Software, San Diego, CA). 
 

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Ethics and dissemination 
This study is approved by the University of Pennsylvania Institutional Review Board (IRB # 829765) and informed 
consent will be obtained prior to enrollment. The dataset supporting the results of this study will be available in 
the Zenodo research data repository. This study is registered with ClinicalTrials.gov at NCT05330676. 
 
Status and timeline of the study 
Initial pilot testing, staff education, and laboratory calibration testing began recruitment of the participants began 
on September 1, 2020. The study is actively enrolling subjects at the time of this publication. Preliminary analysis 
of microcirculation images, mitochondrial respiration, and VVFDs will be conducted in 2022.  
 
SAFETY MANAGEMENT 
 
Clinical Adverse Events 
Clinical adverse events (AEs) will be monitored throughout the study.  
Adverse Event Reporting 
Since the study procedures are not greater than minimal risk, SAEs are not expected. If any unanticipated 
problems related to the research involving risks to subjects or others happen during the course of this study 
(including SAEs) these will be reported to the IRB AEs that are not serious but that are notable and could 
involve risks to subjects will be summarized in narrative or other format and submitted to the IRB at the time of 
continuing review.  
 
Safety considerations 
The Principal Investigator will be primarily responsible for the study conduction throughout protocol completion. 
Research personnel will be required to report any relevant protocol deviations or research related adverse events 
to the Principal Investigator. Risks associated with sublingual IDF imaging are low and no identifiable adverse 
events have been previously linked to sublingual IDF imaging. 
 
 

Protocol date: 6/1/2020 
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STUDY ADMINISTRATION 
 
Data Collection and Management 
Data abstracted from the medical record will be managed and stored using the research-focused electronic 
data capture system REDCap (Research Electronic Data Capture) under an agreement with the software’s 
development consortium, led by Vanderbilt University. We will use the REDCap server located at HUP.  
Data will be managed and stored using the research-focused electronic data capture system REDCap, under 
an agreement with the software’s development consortium, led by Vanderbilt University. REDCap supports two 
secure, web-based applications designed exclusively to support data capture for research studies. 
  
REDCap as implemented at HUP includes daily destructive database backup files that are stored on the 
database server and are deleted only after successful backup of the entire database to file.  Data and backups 
are stored in the HUP Research Information Systems Storage Area Network (SAN).  Access to the SAN 
directories where data are stored will be limited to Research Information Systems personnel, with 
authentication performed using HUP’s enterprise Active Directory service. 
 
Each study patient will be given a study identification number (study ID).  Only the study ID will be recorded on 
the CRF.  A master list linking the study ID to identifying information will be kept separate from the CRF and 
stored as a locked Excel file on the hospital’s secure servers.  Dates (e.g., date of birth, date of hospitalization, 
date of study enrollment) will be recorded in REDCap in order to determine age, length of stay, etc.  However, 
only coded data will be exported from REDCap to be used for analysis of study results.  We will use the 
automated feature within REDCap to shift all dates by a value between 0 and 364 days.  Date shifting is 
important for de-identification because dates are identifiers that can be used for identifying an individual and 
thus possibly exposing confidential personal information.  Date shifting will prevent any dates from being used 
as identifiers while preserving the interval between dates so that age can be calculated.  Date shifting will be 
used to de-identify data prior to sharing with colleagues at Penn.    Access to PHI (i.e the master list and dates 
in REDCap) will be limited to the PHI and key study personnel at HUP.  Those not involved in data entry will 
not have access to view PHI (i.e., dates) within REDCap (using REDCap’s security features to limit data 
access) and will be limited to viewing coded data and shifted dates only.  If paper versions of the CRF are 
needed (e.g., temporary loss of access to REDCap), these will be kept in a locked cabinet in the PI’s office and 
will be coded using the study ID.  Only study personnel authorized by the IRB will have access to study-related 
data and files.   
The password-protected master list linking patient identifiers to the study ID will be retained indefinitely. 
 
Data sources 
This study will in no way alter or interfere with the clinical care of any shock or control subject as determined by 
the medical care team.  All clinical data will be abstracted from the paper or electronic (EPIC and Sunrise) 
medical record.  To assist in this effort, we will perform manual review of the medical record for each study 
patient. At HUP, each data collector and the Data Coordinator have completed extensive initial training and 
monitor data quality quarterly, including inter-rater reliability.  
Confidentiality 
All data and records generated during this study will be kept confidential in accordance with Institutional 
policies and HIPAA on subject privacy and that the Investigator and other site personnel will not use such data 
and records for any purpose other than conducting the study. Safeguards to maintain subject confidentiality are 
described under Data Collection and Management. 
Confidentiality of the data will be ensured by keeping a master list containing patient identifiers and study ID 
separate from data forms (paper and electronic).  The master list will be kept in a locked file on the password-
protected computer of the PI, with a paper copy stored in a locked file cabinet in the PI’s office.  All study-

Protocol date: 6/1/2020 
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related files will be password-protected.   Patient identifiers, including the master list, will be destroyed within 
three years after publication.   
For data security, a copy of all password-protected files will be maintained on the Investigator’s office computer 
(which itself is password-protected), with the original in one of the Hospital’s secure servers. 
No identifiable data will be used for future study without first obtaining IRB approval. The investigator will obtain 
a data use agreement between provider (the PI) and any recipient researchers (including others at HUP) 
before sharing a limited dataset (dates and zip codes). Samples processed in the Metabolomics lab and the 
will be coded with a study ID only and PHI will not be shared.   
 
Regulatory and Ethical Considerations 
Data and Safety Monitoring Plan 
The systems for storing and backing up data were described above.  The principal investigator will be 
responsible for monitoring data collection and the safety of all study measurements.  Given the minimal risks 
and observational nature of the study, we do not require an independent medical monitor of safety.   
 
Risk Assessment 
The risks for patients participating in this study are not greater than minimal risk and include those related to 1) 
blood collection for study measurements and 2) breach of confidentiality. 
Since we will limit the volume of blood collection to less than 3 mL/kg or 50 mL and no more than two blood 
collections in one week, this falls under the provision of “not greater than minimal risk.”  For any patient whose 
clinical condition might be adversely affected by removal of the stated blood volume for this study (e.g., 
patients with significant anemia or compromised cardiac output), we will discuss further limiting the volume of 
blood withdrawn for research purposes with the patient’s attending physician.  Any patient concurrently 
enrolled in another study for who enrollment in this study would mean the combined blood volume collection 
would exceed IRB regulations will be excluded from participation in this study.  Finally, since patients with 
shock typically undergo multiple blood draws per day and often have central and/or arterial vascular access 
devices, whenever possible blood sampling for study purposes will be timed with other clinically indicated 
blood draws or catheter access in order to limit pain from venopuncture, arterial puncture, and access to 
indwelling vascular catheters.  Only caregivers with expertise and training in venopuncture, arterial puncture, or 
access of indwelling catheters will be permitted to collect blood specimens for this study. 
In the rare cases where phlebotomy is performed for study purposes only, attempts will be made to mitigate 
patient discomfort by using topical anesthestics, such as EMLA.  Also, risks will be minimized by limiting the 
number of attempts to obtain blood to 2 per patient per day and only individuals skilled at performing 
phlebotomy will be permitted to attempt. 
To minimize a potential breach of patient confidentiality, CRFs will be coded with a study ID, all data will be 
password-protected, and the safeguards to ensure confidentiality as described. 
Potential Benefits of Study Participation 
There are no direct benefits to patients as a result of participation in this study. 
The indirect benefits of this study are clear.  The knowledge gained through participation may lead to improved 
diagnosis, prognosis, and therapy for adults with life-threatening shock and will advance the state of 
knowledge about progressive organ dysfunction in postoperative shock.  The data gathered from this study will 
be used to inform subsequent studies in shock. 
Risk-Benefit Assessment 
Given the minimal risk profile associated with this study, the overall benefits to science, medicine, future 
generations of postoperative individuals, and society as a whole outweigh the risks. 

Protocol date: 6/1/2020 
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Informed Consent/Assent and HIPAA Authorization 
For patients who meet criteria, the patient or next of kin/power of attorney will be approached for study 
enrollment.  The PI, co-investigators, or trained staff members will engage them in a discussion regarding 
reasons for the study, the study procedures, and the risks and benefits and answer all questions.  Due to the 
often critical nature of the patients’ conditions in the inpatient floor, preoperative clinic, or intensive care unit, 
this discussion may take place at the patient’s bedside or in an alternative location (e.g., family conference 
room) at the next of kin or power of attorney’s  option and the study investigator’s discretion.  Also, since it is 
common for the next of kin or power of attorney to be away from the bedside, we anticipate the need to seek 
consent over the phone for many patients.  Regardless of the where this discussion takes place (i.e., in person 
or via telephone), all reasonable safeguards to ensure patient privacy will be taken.  Next of kin/power of 
attorney will be given sufficient (i.e., up to several hours) to make a decision to participate in this study. 
 
Following the above conversation seeking informed consent from each patient or their surrogate decision 
maker and ensuring that this party understand the study objectives and procedures, we will obtain written 
permission on the combined consent-HIPAA authorization form for all patients for whom the parent/guardian is 
physically present.  If the next of kin/power of attorney is not physically present, we seek a waiver of 
documentation of consent and HIPAA Authorization to obtain verbal consent, which will be documented on the 
standard consent form that will be read to subjects with a verbal consent documentation page. At any point 
during the study time period, patients will be able to withdrawal themselves from the study, as critically ill 
patients may regain capacity to make decisions regarding the study. 
 
We will provide a copy of the combined consent-HIPAA authorization document to the party and patient and 
will write a note in the patient’s medical record documenting the informed consent discussion. 
If the next of kin/power of attorney consents to participate in the study, the next of kin/power of attorney and 
patient will be notified that any remaining blood samples may be stored in a coded fashion for possible use in 
future research.  Next of kin/power of attorney and patients will have the opportunity to decline use of 
remaining blood samples for future research.  All stored blood samples will be labeled only with a coded study 
identification number that will be linked to patient identifiers via a master list kept separate from these samples 
in the PI’s locked electronic and/or paper files. 
 
Informed consent process for non-English speakers 
For non-English speaking next of kin/power of attorney and patients who speak fluent Spanish, Italian, 
Portuguese, Korean, Haitian Creole, Vietnamese, Chinese, Russian, and Arabic, an interpreter will assist in 
the informed consent discussion.  The interpreter will be fluent in both English and the native language of 
the patient (next of kin/power of attorney) and may serve as the witness.  Informed consent will be 
documented using the HUP standard short-form consent document in the appropriate native language and 
on a Study Summary Document written in English. 
Speakers of languages other than English, Spanish, Italian, Portuguese, Korean, Haitian Creole, 
Vietnamese, Chinese, Russian, and Arabic will not be approached for informed consent until an 
appropriately translated consent form can be obtained (which may be submitted as a separate amendment). 
 
Payment to Subjects/Families 
No monetary or physical reimbursement will be provided to patients or their families for participation in this study.  
The cost of all study measurements will be covered by the investigational team. 
 
PUBLICATION 
The method and results of this study are expected to be presented at local and/or national/international 
scientific meetings and to be submitted for publication in a peer-reviewed journal.  Only de-identified aggregate 
data will be published or presented. 

Protocol date: 6/1/2020 
13 
 
 
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Protocol date: 6/1/2020 
15 
LIST OF ABBREVIATIONS (alphabetical order) 
CABG: Coronary artery bypass graft 
C1: Complex 1 
CII: Complex II 
CIII: Complex III 
CIV: Complex IV 
CI: Cardiac index 
CO: Cardiac output 
CPB: Cardiopulmonary bypass 
CRF: Case report form 
CVP: Central venous pressure 
DO2: Rate of oxygen delivery 
ERAS: Early recovery after surgery 
euroSCORE II: European System for Cardiac Operative Risk Evaluation 
IDF: Incident dark field  
LFTs: Liver function tests 
MAP: Mean arterial pressure 
MFI: Microcirculatory flow index 
MHI: Microcirculatory heterogeneity index 
PAP: Pulmonary artery pressure 
PBMCs: Peripheral blood mononuclear cells 
PPV: Proportion of perfused vessels 
PVD: Perfused vessel density 
RBCs: Red blood cells 
SOFA: Sequential organ failure assessment 
STS: Society of Thoracic Surgeons 
SUIT: Substrate-uncoupler-inhibitor titration 
SvO2: Central venous oxygen saturation 
TVD: Total vessel density 
VO2: Rate of oxygen consumption 
VVFDs: Ventilator and vasopressor-free days