Utilization of Confocal Microscopy During Cardiac Surgery
Phase: PHASE1
Conditions: Atrial Septal Defect
Sponsor: Aditya Kaza

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Study Protocol 
Protocol Title: Utilization of Confocal Microscopy during Cardiac Surgery 
Document Date: October 19, 2018 
IRB Approval Date: October 31, 2018 
NCT Number: NCT03189134  

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Utilization of Confocal Microscopy during Cardiac Surgery 
A. 
Specific Aims/Objectives  
This is a pilot study, exploring the safety and feasibility of using diluted fluorescein sodium with 
fiberoptic confocal microscopy during cardiac surgery.   
 
The primary aim of this study is to evaluate the safety of using diluted fluorescein sodium 
during cardiac surgery.   
 
A secondary aim of this study is to evaluate the feasibility of using this imaging modality.  
Feasibility will be evaluated in two ways: logistically and technologically. 
o Logistical feasibility will be evaluated by measuring increased time on bypass, rate of 
enrollment, and the ability to incorporate this imaging modality into standard OR 
procedures. 
o Technological feasibility will center on the ability to capture quality image files.  A 
quality image file is defined as an image file where a rater can visualize the 
distinguishable features of the tissue type (cardiac tissue, conductive tissue) in order to 
classify that tissue type correctly. 
 
B. 
Background and Significance  
 
Congenital heart disease is the most common type of birth defect. Approximately 32,000 new cases of 
congenital heart disease occur in the US every year, and there are approximately 1.5 million new 
cases annually worldwide.1, 2 More than 20,000 pediatric cardiac operations are performed each year 
in the US.3 Over the past two decades, emphasis has been on the complete repair of the congenital 
cardiac defects in infancy. This paradigm shift has improved the surgical outcomes and 
neurodevelopmental progress of these infants. Without surgical intervention, these defects are 
uniformly lethal.4 However, there are several complications associated with surgical intervention, 
including dysfunction of sino-atrial and atrio-ventricular conduction pathways. Despite the complexity 
and individual variations in cardiac conductive pathways, tissue discrimination during surgery is 
currently limited to the use of anatomic landmarks and measurements of local electrical activity, so 
accurate surgical intervention is therefore challenging and risky. In addition, there are only sparse 
data on the location and development of these pathways in pediatric hearts with congenital defects.5 
One of the perioperative complications that can be induced during these procedures is complete heart 
block purportedly associated with interruption of cardiac conduction pathways, which can occur due to 
damage to conductive tissues from clamping, suturing, or other procedures. Complete heart block 
contributes significantly to increased morbidity and mortality.6 Once complete heart block has 
occurred and there has been no recovery by the patient, a permanent cardiac pacemaker must be 
implanted.7 This creates a lifetime dependence on the pacemaker along with the associated lead and 
generator changes.8 A study involving 497 patients over a 22-year period showed that lead failure 
occurred in 155 leads (15%) and 115 patients (23%), with a higher incidence of lead failures in 
pediatric patients.9 With more complex procedures, the risk of complete heart block or sinus node 
dysfunction increases accordingly. Lifetime costs and morbidity associated with pacemaker 
implantation in pediatric patients are very high.8 The economic burden of this rhythm management 
therapy is significant, especially when considering the average lifespan of these young patients.10 
There is also a societal burden, as evidence points to long-term morbidity associated with surgical 
correction of congenital heart defects.11 Despite the significant improvement in surgical results, there 
is still a critical barrier that needs to be overcome, which is to avoid preventable causes of conduction 
defects. 
 
The Cellvizio 100 Series System with Confocal Miniprobes is a confocal laser system with fiber optic 
probes that has been approved by the FDA to allow real-time imaging of the internal microstructure of 
tissues in anatomical tracts. This system is capable of acquiring and displaying microscopic images 

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directly after tissue contact with a rate of up to one image every 5 milliseconds (200 images per 
second). Preliminary studies (described in more detail below) suggest that image data obtained by 
this system allows for discrimination of the cardiac conduction system. We hope to develop a system 
for intraoperative identification of tissue of the conduction system, which could potentially decrease 
the incidence of preventable causes of conduction delays in the operating room. Reducing the 
damage to the conduction system tracts may preserve autonomic nervous activity and lead to 
improved short and long term outcomes for these patients.12 Furthermore, this technology could be 
utilized not only for reconstructive pediatric heart surgery, but also for other corrective surgeries where 
detailed tissue discrimination would improve patient outcomes. 
 
C. 
Preliminary Studies  
 
In a recently published study, we established an approach based on fluorescent labeling, conventional 
confocal microscopy, and image analysis to characterize cardiac tissue types.13 Tissue preparations 
from adult rat hearts were dissected using anatomical landmarks such as the superior vena cava and 
coronary sinus. The tissue preparations were labeled with 4’,6-diamidino-2-phenylindole (DAPI) to 
identify nuclei, wheat germ agglutinin (WGA) conjugated Alexa 488 to define the extracellular space, 
antibodies for hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) to 
identify nodal myocytes14-17 and sarcomeric α-actinin to identify cardiomyocytes. Conventional 
confocal microscopy of the tissue preparations yielded 3D distributions of the four labels at a high 
resolution.   
 
Using this method, we were able to characterize the microstructure of various cardiac tissue types. 
Atrial working myocardium (AWM) was characterized by a dense and aligned arrangement of 
myocytes that were covered by an epicardial layer with a thickness of 8.26±3.60 µm. Sino-atrial node 
(SAN) was characterized by three distinct layers comprised of epicardium, SAN cells, and subjacent 
myocytes of the AWM. The epicardial and nodal layers had thicknesses of 10.38±4.79 µm and 
10.26±3.33 µm, respectively. SAN cells formed a highly irregular reticulum and had notably smaller 
diameters than myocytes of the AWM. The extracellular space in the SAN layer was also more 
prominent than in AWM and was comprised of oval shaped clearings of variable size. Atrio-ventricular 
node (AVN) and Bundle of His tissue presented a microstructural arrangement similar to SAN. It 
exhibited major differences from AWM tissue, in particular, an irregular reticulum of cells with ample 
extracellular space. The epicardial layer of AVN tissue had a thickness of 2.84±1.25 µm. 
 
Following the investigations using conventional confocal microscopy, we investigated the hypothesis 
that fiberoptic confocal microscopy (FCM) technology could be used to identify the differences in 
tissue microstructure between nodal tissue and working myocardium in living Langendorff perfused rat 
hearts. An aqueous solution of 10 kDa dextran-conjugated Alexa 488 was pipetted to the tissue 
surface. It has previously been shown that dextran-conjugates of this molecular weight penetrate 
endothelial endocardium, endothelial epicardium, and myocardial capillary endothelium in rat, but do 
not penetrate intact cell membranes, and thus label specifically the extracellular space.18 We found 
that FCM images from the dextran-labeled AWM and nodal regions resembled images obtained using 
conventional confocal microscopy.  
 
The FCM images were of high enough quality to visually distinguish tissue types. We also explored 
approaches for automated image analysis of the conventional microscopy and FCM images. We 
evaluated the ability of two methods of 2D texture analysis to discriminate between AWM and nodal 
tissue using images of the extracellular space. The first method measured the texture orientation from 
Fourier transformed images.19 The second method characterized texture orientation from second 
order image moments of image regions.19 Both methods of 2D texture analysis yielded a larger 
magnitude of our defined measure of orientation in AWM in comparison to SAN and AVN tissue. 
 

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In further studies, we demonstrated that fluorescein sodium (Fluorescite®) is an equally reliable label 
of extracellular space. Fluorescite® is approved by the FDA for intravenous administration for use in 
ophthalmic angiography, and its toxicity has been extensively studied.20 Fluorescite® is also used in 
gastrointestinal endoscopic imaging. The incidence of adverse reactions following intravenous 
administration of fluorescein is 1.1-4.8% for retinal fluorescein angiography and 1.4% for 
gastrointestinal endoscopic imaging.21-23 In these studies, adverse reactions including dizziness, 
nausea, vomiting, and transient hypotension have been reported. However, there were no serious 
adverse reactions or deaths. For our purposes, we determined that localized, topical delivery of the 
dye would allow us to use a smaller amount and be equally effective.24 Human examiners were able 
to classify FCM images obtained using fluorescein delivered by dye carrier as either AWM or nodal 
tissue with high degrees of sensitivity (99.2%±0.3) and specificity (98.0%±0.7). In further studies, we 
showed that topical delivery of the dye by pipette or surgical swab was equally effective to the dye 
carrier method, and allowed us to use a concentration of dye that is 1000 times more dilute than what 
is approved by the FDA. 
 
Our research team is currently conducting experiments in sheep, but the data have not been 
published. In these studies, 3 sheep have been anesthetized and placed on cardiopulmonary bypass. 
Between 5-7mLs of 1:1000 diluted Fluorescite® was applied to the epicardium and endocardium, and 
images were obtained using the Cellvizio Imaging system. After the images were obtained, the 
animals were taken off bypass. ECGs were obtained at baseline, after re-perfusing the hearts, and 
after the animals were taken off bypass. The ECGs confirmed that after the procedure, the hearts 
resumed normal baseline cardiac activity. Visual examination of the hearts also confirmed normal 
ventricular function and vigorous activity. Furthermore, preliminary imaging data from these studies 
confirms that we are able to discriminate conduction tissue from the images taken. 
D. Design and Methods  
(1) 
Study Design 
 
We propose a descriptive pilot study to assess the safety and feasibility of using fiber-optic 
confocal microscopy during cardiac surgery to image the endocardium and epicardium. 6 
patients undergoing surgery for closure of atrial septal defect will be enrolled in this study.  
 
All enrolled subjects will receive standard of care for cardiac surgery and the operation will be 
conducted according to routine procedure. After the sternotomy, the subject will be placed on 
cardiopulmonary bypass. After cardioplegic infusion and cardiac arrest, the right atrium will be 
opened to visualize the atrial septal defect.  
 
At this juncture, fluorescein sodium at a concentration of 100ug/ml in sterile saline will be 
applied topically to the myocardium, where it will diffuse into the endocardium and epicardium. 
A total volume of 5ml will be applied. The surgeon will confirm there are no visual indicators of 
an allergic reaction to the fluorescein study drug.  As long as there is no appearance of tissue 
damage, the imaging will proceed. If any signs of allergy or intolerance occur, the dye will be 
immediately flushed out and no images will be obtained. 
 
The imaging probe of the Cellvizio system will then be applied in direct contact to the region of 
interest with minimal pressure for approximately 90 seconds. During this period, Cellvizio 
Software and Viewer will be used to obtain images. The total additional time on bypass will be 
approximately 3 minutes. 
 
Once the imaging procedure has been completed, the heart surface will be washed with saline 
and evacuated using a suction apparatus, to ensure all fluorescein has been removed and will 

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not enter the circulatory system.  After that, the remainder of the operation will proceed in the 
usual fashion. As per standard of care for patients undergoing cardiac surgery, an 
intraoperative echocardiogram will be performed after the subject is separated from bypass. 
 
The expected hospitalization for the Atrial Septal Defect (ASD) is 3-5 days post-operation. It is 
not expected that participation in this study will increase the post-operative hospital stay or 
post-hospitalization recovery.  The principal investigator (PI) and research team will monitor 
clinical labs, electrocardiograms (EKGs), and echocardiograms for any evidence of adverse 
events.  No additional follow-up testing, outside of standard clinical monitoring, are planned. In 
accordance with standard of care, all subjects will receive a pre-discharge EKG and trans-
thoracic echocardiogram.   
 
Image files will be exported out from the Cellvizio system, de-identified, and provided to the 
blinded raters. The image files will be assessed for quality and the ability to discriminate tissue 
types. 
 
(2) 
Patient Selection and Inclusion/Exclusion Criteria  
 
Patients undergoing cardiac surgery for ASD repair at Boston Children’s Hospital will be 
enrolled in this study. Surgery for ASD repair exposes the conduction system of the heart, 
allowing access to the AWM and nodal tissue for image capture by the Cellvizio system. 
Congenital heart defects such as ASD are most often diagnosed and repaired in infants and 
children, which is why conducting this study in a pediatric population is appropriate.  
Additionally, the FCM has a pre-defined field of focus, with a depth ranging from 55 to 65 µm. 
Therefore, this technology is more suited to pediatric hearts, as adult hearts are thicker and 
the conductive tissue may be too deep to be identified using this method. 
 
Inclusion criteria: 
 
Males and females between 30 days and 21 years old 
 
Undergoing elective surgery for closure of isolated secundum atrial septal defect 
 
Both parents attend pre-operative clinic appointment, to provide 2 parent consent, or 
an adult participant (age 18-21) may provide consent for him/herself 
 
Exclusion criteria: 
 
Prior history of adverse reaction to fluorescein sodium 
 
Prior history of renal failure or abnormal renal function 
 
Baseline PR interval > 220 msec or 98% for age 
 
Baseline HR > 98% for age 
 
Underlying genetic syndrome associated with progressive AV block of sinus node 
dysfunction (e.g. Holt-Oram or NKX2.5) 
 
(3) 
Description of Study Treatments or Exposures/Predictors  
 
(3.1) Investigational Agent 
 
Fluorescite® (fluorescein injection, USP) 10% contains fluorescein sodium (equivalent to 
fluorescein 10% w/v) (Alcon). Fluorescite® is commercially available, a supply of which will be 
purchased for the sole purpose of this study. The Boston Children’s Hospital (BCH) research 
pharmacy will be responsible for the receipt and accountability of study agent. The pharmacy 
will maintain accountability logs with quantities received and dispensed, specifying the 
corresponding dates, and the identification code of the study subject. 

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The research pharmacy will dilute 1mL of the study drug into 1L of saline. A 5mL syringe of the 
1:1000 diluted solution will then be dispensed to the OR. Any unused prepared study drug will 
be recorded, and then discarded or destroyed according to Boston Children’s Hospital policy.  
The vial of undiluted Fluorescite® will also be discarded or destroyed according to BCH policy. 
 
(3.2) Device 
 
The Cellvizio 100 Series System and the Gastroflex UHD probe (Mauna Kea Technologies) 
will be used for imaging in this study. This instrument has been approved by the FDA for 
gastrointestinal, urologic, gynecologic, and pulmonary applications. This device will be 
provided by the manufacturer for the purposes of this study, along with the associated control 
and acquisition software. The PI has been thoroughly trained on the use of this hardware and 
software. The cardiac surgery operating room will be responsible for the receipt, 
accountability, and return of the microscope tower and imaging console. The imaging console 
will be stored in the cardiac operating room. The microprobes will be sterilized according to 
standard protocol and stored in a sterile pouch in the operating room. The imaging console is 
password protected and will only be accessed by the PI. 
 
The probe will be used in direct contact with the cardiac tissue with a pressure of 
approximately 0.7 mmHg, which is less than 1% of diastolic blood pressure. The maximum 
laser energy that the system is capable of delivering is 15 mW at 480 nm. This power and 
wavelength has been approved by the FDA for direct in-vivo imaging for various body tissues, 
including gastrointestinal, urogenital, and pulmonary. The proposed application is 
approximately 80% less than the maximum allowable retinal exposure, according to the IEC 
60825-1 standard. 
 
(4) 
Definition of Primary and Secondary Outcomes/Endpoints  
 
Primary Outcome: All adverse events will be collected and relatedness to study participation 
will be determined. Complications will be compared to average complication rates for ASD 
repairs. 
 
Hypothesis: Subjects will not experience a higher rate of adverse events than is expected in a 
clinical setting (<3-4% for all types of adverse events). 
 
Secondary Outcome: The logistical feasibility of conducting FCM imaging during cardiac 
surgery. 
 
Hypothesis: The study procedure will be logistically feasible in terms of added time on bypass 
and the ability to incorporate FCM imaging into standard cardiac surgery OR procedures.  The 
rate of enrollment will be evaluated separately. 
 
Secondary Outcome: The quality of the FCM image files collected, defined as the ability of 
observers to distinguish features and discriminate between working myocardium and nodal 
tissue.  
 
Hypothesis: Blinded raters will be able to use the FCM image files to distinguish features and 
discriminate between working myocardium and nodal tissue. 
 
(5) 
Data Collection Methods, Assessments, Interventions and Schedule (what 
assessments performed, how often)  

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Standardized demographic and clinical assessments of all study participants will be performed 
via medical record review prior to and after surgery until the subject is discharged from the 
hospital. Images will be obtained during surgery and saved using Cellvizio Software and 
Viewer. The image files will be de-identified and provided to blinded raters for analysis. 
Medical records of enrolled subjects will be reviewed post-surgery while the subject is inpatient 
in the hospital.  Adverse events will be abstracted from the record and recorded on a CRF and 
input into an InForm database. 
 
(6) 
Study Timeline 
 
With several patients eligible weekly, it should be feasible to complete subject recruitment, 
enrollment, and follow-up within 6 months after study activation. Lead time will be needed to 
secure FDA and BCH Institutional Review Board (IRB) approval. 
E. Adverse Event Criteria and Reporting Procedures  
(1) 
Specification of Safety Parameters 
 
Comprehensive assessments of any apparent toxicity experienced by the study subject will be 
performed throughout the course of the study. All safety events experienced by study subjects 
for the duration of the study will be documented. The types of adverse events that must be 
reported and procedures for appropriately recording, grading and reporting are outlined in this 
section. 
 
(2) 
Definition of an Adverse Event (AE) or Medical Event 
 
According to FDA regulations, an adverse event (AE) is any undesirable, noxious, or 
pathological change compared to preexisting conditions occurring to or in a study subject 
enrolled in a clinical trial, whether or not the event is considered related to the test drug. This 
includes the time periods during which no medication is administered to a study subject, such 
as run-in or follow-up periods. An adverse event is further defined as any change in structure 
(signs) or function (symptoms) of the body that occurs while the study subject is receiving 
study drug/placebo. AEs include the following types of changes: 
 
 
Suspected adverse drug reactions, which are any adverse events for which there is a 
reasonable possibility that the drug caused the adverse event. A reasonable possibility 
implies that there is evidence that the drug caused the event. 
 
 
Other medical experiences regardless of their relationship to the test drug, such as 
injury, surgery, accidents, extensions of symptoms or apparently unrelated illnesses, 
and significant abnormalities in clinical laboratory values, physiological testing, or 
physical examination findings. 
 
 
(3) 
Definition of Serious Events (Serious Adverse Events, Serious Suspected 
Adverse Reactions or Serious Adverse Reactions)  
 
A serious adverse event including a serious suspected adverse reaction or serious adverse 
reaction as determined by the Investigator or the sponsor is any event that results in any of the 
following outcomes: 
 

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1. Death. This includes any death that occurs during the conduct of a clinical study, 
including deaths that appear to be completely unrelated to the test drug (e.g., a car 
accident). If a study subject dies during the study and an autopsy is performed, the 
autopsy results will be attached to the study subject’s Case Report Form (CRF). 
Possible evidence of organ toxicity and the potential relationship of the toxicity to the 
test drug are of particular interest. The autopsy report should distinguish the 
relationship between the underlying diseases, their side effects, and the cause of 
death. 
 
2. Life-threatening adverse drug experience. This includes any AE during which the study 
subject is, in the view of the investigator, at immediate risk of death from the reaction 
as it occurred. This definition does not include any event that may have caused death if 
it had occurred in a more serious form. For example, drug-induced hepatitis that 
resolved without evidence of hepatic failure would not be considered a life-threatening 
event, even though drug-induced hepatitis can be fatal. 
 
3. Persistent or significant disability or incapacity or substantial disruption of the ability to 
conduct normal life functions. 
 
4. Inpatient hospitalization or prolongation of existing hospitalization, except for admission 
for elective surgery. 
 
5. Important medical event that may not result in one of the above outcomes, but may 
jeopardize the health of the study subject or require medical or surgical intervention to 
prevent one of the outcomes listed in the above definition of serious event.  
 
(4) 
Definition of Unexpected Adverse Events 
 
An adverse event is “unexpected” when its nature (specificity) or severity is not consistent with 
applicable product information, such as safety information provided in the package insert, the 
investigational plan, the investigator’s brochure or the protocol. 
 
In addition, an adverse event is “unexpected” when not one of the most common 
complications that typically arise post-operation for ASD repair such as: rhythm disturbances, 
arrhythmia, pericarditis, cardiac tamponade, bleeding, residual holes requiring a re-operation, 
transfusion associated diseases, stroke, and brain damage. These adverse events will be 
considered expected and unrelated to the study drug.    
 
(5) 
Toxicity Grading 
 
We will assign toxicity grades to indicate the severity of adverse experiences and toxicities. 
Toxicity grading consistent with NCI-CTCAE v 4.03 will be used for this protocol. The purpose 
of using the NCI-CTCAE system is to provide standard language to describe toxicities and to 
facilitate tabulation and analysis of the data and assessment of the clinical significance of 
treatment-related toxicities. 
 
Adverse events not included in the NCI-CTCAE listing will be recorded and graded 1 to 5 
according to the General Grade Definition provided below:  
 

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Grade 1 
Mild 
Transient or mild discomforts (< 48 hours), no or minimal 
medical intervention/therapy required, hospitalization not 
necessary (non-prescription or single-use prescription therapy 
may be employed to relieve symptoms, e.g., aspirin for simple 
headache, acetaminophen for post-surgical pain). 
Grade 2 
Moderate 
Mild to moderate limitation in activity, some assistance may be 
needed; no or minimal intervention/therapy required, 
hospitalization possible.   
Grade 3 
Severe 
Marked limitation in activity, some assistance usually required; 
medical intervention/therapy required, hospitalization possible.   
Grade 4 
Life-threatening 
Extreme limitation in activity, significant assistance required; 
significant medical/therapy intervention required, 
hospitalization, or hospice care probable. 
Grade 5 
Death 
Death 
 
 
 
For additional information and a printable version of the NCI-CTCAE v. 4.03 manual, consult 
the NCI-CTCAE website, http://ctep.cancer.gov/reporting/ctc.html. 
 
(6) 
Guidelines for Determining Causality of an Adverse Event 
 
The Investigator will use the following question when assessing causality of an adverse event 
to study drug:  Is there a reasonable possibility that the drug caused the event? An affirmative 
answer designates the event as a suspected adverse reaction. 
 
If there is any doubt as to whether a clinical observation is an AE, the event should be 
reported.  All AEs must have their relationship to study product assessed using the terms 
related or not related.  In a clinical trial, the study product must always be suspect.  To help 
assess, the following guidelines are used: 
 
 
Related – Related means there is evidence to show a causal relationship between the 
study product and the adverse event. 
 
 
Possibly Related – There is a reasonable possibility that the study product caused the 
adverse event. Reasonable possibility means that there is evidence to suggest a 
causal relationship between the study product and the adverse event. 
 
Specific adverse events considered related or possibly related to study participation 
have been identified as: myocarditis or ventricular dysfunction, as measured by 
ejection fraction and fractional shortening.  These will be reported to the FDA in an IND 
Safety Report, per discussion with the FDA.   
 
Specific ECG and rhythm abnormalities requiring DSMB review prior to enrollment of 
next patient: 
1.    AV block > the a PR interval > 220 msec lasting more than 2 hours post 
completion of cardiopulmonary bypass.  This does not include transient 
Wenkebach with sleep/rest/hypothermia.   
2.    Need for atrial pacing or junctional rhythm lasting more than 6 hours post 
completion  
3.    Hypotension or ventricular dysfunction requiring pressor support > 5mcg/kg/min of 
dopamine more than 4 hours post completion of cardiopulmonary bypass.  

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 
Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event, and/or there is a more plausible explanation for the adverse 
event than administration of the study product. 
 
(7) 
Recording and Reporting Procedures  
 
Reporting Adverse Events and Serious Adverse Events 
 
All AEs will be followed until the time the event is resolved or medically stable, or until subject 
is discharged from hospital, whichever comes first. AEs may be discovered through any of 
these methods: 
 
 
Observing the subject 
 
Questioning the subject, which should be done in an objective manner 
 
Receiving an unsolicited complaint from the subject 
 
Review of medical records/source documents 
 
All AEs must be recorded accurately on the Adverse Event log for each study subject. The 
date of onset and duration of the AE, grade, whether it was expected or unexpected, action 
taken, date of resolution (if applicable) and outcome of the event must be recorded. The 
investigator will treat subjects experiencing adverse events appropriately and observe them at 
suitable intervals until their symptoms resolve or their status stabilizes or they are discharged 
from the hospital.  
 
SAEs and unexpected problems will be reported promptly to BCH IRB according to local 
regulations and guidelines. 
 
The PI and delegates are responsible for notifying the DSMB chair of all SAEs. DSMB panel 
will evaluate the relationship of the SAE to the underlying disease.  The PI, in cooperation with 
the DSMB panel, will determine whether the SAE is unexpected in nature and make a 
recommendation for any further action. The primary consideration is the study subjects’ safety. 
 
(8) 
Regulatory Reporting 
 
The PI will report events that are both serious and unexpected and that are associated with 
Study Agent/Intervention to the FDA and other applicable health authorities within the required 
timelines as specified in 21 CFR 312.32: fatal and life threatening events within 7 calendar 
days (by phone /fax/electronic communication) and all other serious adverse events in writing 
within 15 calendar days.  All serious events designed as “not associated” to Study 
Agent/Intervention, will be reported to the FDA at least annually. 
 
Given the low dose and method of delivery of fluorescein sodium, serious adverse events 
related to the study drug are not anticipated. Subjects will be monitored at the time of dye 
application to observe for signs of allergy or intolerance. If any signs of allergy or intolerance 
occur (as evidenced by discoloration of tissue or other signs of allergic reaction), the dye will 
be immediately flushed out and no images will be obtained. As per standard of care for 
patients undergoing cardiac surgery, subjects will receive an intraoperative echocardiogram 
after being separated from bypass, and will be monitored closely post-surgery. Before being 
discharged, all subjects will receive an electrocardiogram and trans-thoracic echocardiogram, 
in accordance with standard of care.  

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F. Data Management Methods  
Study specific case report forms (CRFs) will be completed and the case report forms will be stored in 
individual subject binders. Data will be transcribed to a secure research data management system 
(InForm database). All hardcopies will be kept in a locked file cabinet accessible by authorized study 
staff only. Subject confidentiality will be maintained by recording subject data using a unique subject 
identifier. Identifiable linking data, such as contact information and medical record numbers, will be 
recorded and stored separately from the clinical study data.  
 
FCM image files obtained for the study will be stored on a password-protected hard drive connected 
to the Cellvizio system. The image files will be exported, labeled only with the subject’s study ID 
number, and provided to the blinded raters for analysis. 
 
G. Quality Control Method  
All study related tasks will follow Good Clinical Practice guidelines. A manual of operations (MOO) will 
be developed to detail the specific procedures to be conducted and data to be collected. Any 
significant deviations or exceptions to the protocol will be reported to the IRB and FDA in addition to 
being discussed among the investigators, and the PI will develop and implement a plan of action as 
needed (e.g. modification to MOO, study amendment, etc.). The PI will ensure the retention of all 
necessary records and reports for a minimum of 2 years after the discontinuation of the IND.  The 
FDA will be allowed access to these records.  A record of accountability of all investigational drug 
dispositions will be maintained, including batch and lot numbers and storage records. 
 
H. Data Monitoring 
 
Monitoring and auditing procedures as outlined in the Monitoring Plan will be followed to ensure that 
the entire study is conducted, documented, and reported in accordance with the IRB approved 
protocol, the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) 
Guidelines, and applicable regulatory requirements of Boston Children’s Hospital. 
 
Monitors will review and verify protocol compliance with particular focus on AE/SAE reporting, CRF 
data, source documentation, informed consents, and any other study-related documentation, including 
review of hospital pharmacy procedures, drug accountability documentation, and drug storage 
facilities and records. 
 
I. Data Analysis Plan  
 
The FCM image files will be provided post-surgery to blinded raters, who will attempt to categorize the 
image files as either myocardium or nodal tissue. The raters will note if the image quality is not high 
enough to discriminate tissue types.  
J. Statistical Power and Sample Considerations  
This is a single site, descriptive, pilot study, so no power calculations have been performed.  Six 
subjects will provide enough images to determine the feasibility of a larger randomized trial, to be 
performed in the future. 
K. Study Organization  
This is a single site pilot study.  Dr. Kaza will perform all study drug administration and image 
acquisition.  BCH research team members will assist in data abstraction and analysis. 

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L. References 
1. 
Moller JH, Taubert KA, Allen HD, Clark EB, Lauer RM. Cardiovascular health and disease in 
children: Current status. A special writing group from the task force on children and youth, 
american heart association. Circulation. 1994;89:923-930 
2. 
Hoffman JI. Congenital heart disease: Incidence and inheritance. Pediatr Clin North Am. 
1990;37:25-43 
3. 
Moodie DS. Adult congenital heart disease. Curr Opin Cardiol. 1994;9:137-142 
4. 
Gross GJ, Chiu CC, Hamilton RM, Kirsh JA, Stephenson EA. Natural history of postoperative 
heart block in congenital heart disease: Implications for pacing intervention. Heart Rhythm. 
2006;3:601-604 
5. 
Anderson RH, Ho SY, Becker AE. The surgical anatomy of the conduction tissues. Thorax. 
1983;38:408-420 
6. 
Wolff GS, Rowland TW, Ellison RC. Surgically induced right bundle-branch block with left 
anterior hemiblock: An ominous sign in postoperative tetralogy of fallot. Circulation. 
1972;46:587-594 
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