Trial of a Topical Nitroglycerin Challenge to Detect Reversible Microcirculatory Dysfunction in Patients With Circulatory Shock.
Conditions: Circulatory Shock, Cardiovascular Shock
Sponsor: University of Pennsylvania

CLINICALTRIALS.GOV 
Title: 
Physiologic 
Effect 
of 
Topical 
Nitroglycerin 
on 
Microcirculation Capacity in Patients With Circulatory Shock. 
NCT: NCT05102734 
 
PI Name: John C. Greenwood, MD 
Contact Email: john.greenwood@pennmedicine.upenn.edu 
 
 
Date: June 1, 2021

Study Protocol and Statistical Analysis Plan 
Patient selection 
Adult patients (age>18 years) receiving elective cardiac surgery requiring cardiopulmonary 
bypass were screened for eligibility from August-October 2021. Written informed consent was 
obtained from eligible patients prior to surgery. Patients were excluded if they had a nitroglycerin 
allergy, were taking oral phosphodiesterase inhibitors, were unable to tolerate microcirculatory 
flow image acquisition or did not have a pulmonary artery catheter for continuous cardiac output 
monitoring after surgery. Patients only received the study drug if they met the clinical criteria for 
circulatory shock, defined as having postoperative vasopressor-dependent hypotension or low 
cardiac output requiring inotropic support, with signs of end-organ injury (capillary refill time > 3 
seconds, lactate > 2, SvO2 < 60%). A second cohort of patients were enrolled to serve as a 
preoperative control, which were matched to the experimental group for age, co-morbidities, and 
planned operation. Study data were collected and recorded using a well-established clinical 
database tool (REDCap, Vanderbilt University, Nashville, TN) hosted at the University of 
Pennsylvania1,2. 
 
Study design and drug administration 
This investigator-initiated, open-label study was conducted at the Hospital of the University of 
Pennsylvania, an urban quaternary academic medical center. The study was approved by the 
University of Pennsylvania’s institutional review board, registered with ClinicalTrials.gov 
(NCT05102734), and conducted in accordance with the principles of the Declaration of Helsinki. 
Sample size was determined based on an anticipated 30% increase in TVD, and setting a one- 
sided α of 0.1, and ß of 0.8. A 1% topical nitroglycerin solution was prepared immediately prior to 
enrollment by reconstituting 400 mcg of nitroglycerin (Pfizer Pharmaceuticals, New York, NY, 
USA) in 4mL of sterile water within a dropper bottle that would administer the solution in 0.05mL 
aliquots. Two drops (0.1 mL) of the 1% nitroglycerin solution (5 mcg or 2.27*10−2 μmol per drop) 

were applied to the sublingual space after obtaining baseline post-operative microcirculation 
images. A microdosing (approximately 1/50th of the lowest therapeutic sublingual dose) was 
administered to avoid any systemic effects. 
 
Microcirculatory imaging and analysis 
Sublingual microcirculation imaging was performed using HVM (CytoCam, Braedius Medical BV, 
the Netherlands) in the preoperative staging area (for controls) or within 2 hours of arriving to the 
intensive care unit after surgery by a trained member of the investigative team (JCG or FMT)3. 
The sublingual microcirculation was measured at 3 time points: baseline, 3 minutes post- 
nitroglycerin, and then 30 minutes later. Images were obtained by gently placing the 
videomicroscope under the subject’s tongue until an adequate view of the microcirculation was 
obtained. A series of six second video clips (120 frames) duration were taken at each time point 
with attention to quality factors, especially the absence of pressure artifact, excess saliva, and 
proper location in accordance with the accepted consensus for microcirculation analysis. Image 
quality was assessed using the 6-factor Massey quality score, which scores each video for 
appropriate illumination, duration, focus, content, stability, and pressure. Images were only 
included in the final analysis if the Massey quality score was < 10 4. Acceptable clips were 
deidentified and coded for analysis after enrollment was complete. 
 
Microcirculation videos were exported using commercial software (CCTools 2, Braedius Medical 
BV, the Netherlands) and manually analyzed using the validated Automated Vascular Analysis 
software (AVA 3.2; Microvision Medical B.V., the Netherlands). Video analysis was performed by 
trained investigators (JCG and FMT) who were blinded to the conditions of the subject. Only 
microvessels < 20 µm in diameter were included in the calculation of TVD and PVD. 

Red blood cell flow velocity was calculated within appropriate venules 20-30 µm in diameter by 
manually measuring the slope of individual RBC movement within software generated space-time 
diagrams 5. Microcirculatory function was quantified according to the current best practice 
guidelines for microcirculation imaging5. Blood flow within each microvessel was graded using a 
semiquantitative scale, based on the vascular flow pattern ranging from 0-3 (0=no flow, 
1=intermittent, 2=sluggish, or 3=continuous flow). Vessel perfusion was dichotomized as 
nonperfused (no flow or intermittent flow) or perfused (continuous or sluggish) for the calculation 
of PPV and PVD. 
 
Individual pharmacodynamic response to the nitroglycerin challenge 
An exploratory analysis was planned to identify the incidence of a patient specific 
pharmacodynamic response to the nitroglycerin challenge. A PD response threshold was set as 
a post-administration PVD > 1.8 standard deviations from the baseline, which was calculated by 
averaging the intra-patient variance between the three baseline PVD video sequences, which was 
then pooled across the entire cohort. This 1.8SD threshold was set to achieve 90 percent 
confidence interval (assuming a one-sided alpha, since there was only an anticipated increase in 
PVD). This method has been used in previous Phase 0 trials to provide a statistically rigorous 
threshold for individualized PD responses to novel therapeutic interventions6. 
 
Patient data and safety monitoring 
Subject demographics, pre-operative risk score (euroSCORE II) and medical history were 
collected during initial screening. Cardiac index (CI), central venous pressure (CVP), pulmonary 
artery pressure (PAP), and mixed venous oxygen saturation (SvO2) were continuously monitored 
by pulmonary artery catheter (Edwards Lifesciences LLC, Irvine, CA, USA). Arterial blood 
pressure was measured continuously using a standard invasive arterial line. Systemic 
hemodynamic data, vasopressor infusion doses, and mixed venous oxygen saturation were 

recorded at baseline then every 3 minutes for 30 minutes after application of the topical 
nitroglycerin solution7. The vasopressor-inotrope score (VIS) was calculated to summarize the 
degree of post-operative vasoactive support was required at the time of the study8. 
Norepinephrine equivalents were calculated for each patient at each time point9. 
 
Availability of data and materials 
The dataset supporting the results of this report is available via the Zenodo research data 
repository10. 
 
Statistical Analysis 
Data normality was assessed using the D’Agostino-Pearson omnibus normality test. Global 
hemodynamic and microcirculation variables are reported as mean ± SD. Variables that were not 
normally distributed are reported as median with interquartile range [25th – 75th percentiles]. 
Student t-test was used to test for differences between normally distributed variables. Mann- 
Whitney U test was used to analyze non-normal data. Repeated measure one-way ANOVA with 
Tukey’s post-hoc testing was used to assess for changes in microcirculatory function during the 
nitroglycerin challenge. Inter-rater reliability between coders was assessed in 10% of the videos 
using a Bland-Altman plot. Statistical analysis was conducted using Prism v 8.0 (Graph-Pad 
Software, San Diego, CA). Statistical significance was assumed at p < 0.05. 

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nitroglycerin challenge to detect reversible microcirculatory dysfunction in patients with 

circulatory shock. Zenodo; 2021 Dec 9 [cited 2021 Dec 8]; Available from: 
https://zenodo.org/record/5768720