Conditions: Hemostasis
Keywords: Fibrinogen Concentrate FC Cryoprecipitate Transfusion in vivo Open-heart surgery Cardiopulmonary Bypass Fibrin Network Structure
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 36
Healthy volunteers: no

Brief summary:
This primary aim of this study is to compare the in vivo effects of fibrinogen concentrate and cryoprecipitate on the neonatal fibrin network after surgery with cardiopulmonary bypass to develop effective and safe strategies for managing coagulopathies in neonates.

Detailed description:
This study is a prospective, randomized control trial comparing two different sources of fibrinogen on clot kinetics (degradation and structure) in post-CPB coagulopathy in neonates undergoing cardiac surgery. The two sources of fibrinogen include the blood product, cryoprecipitate, and a blood product alternative, fibrinogen concentrate. Cryoprecipitate is an allogenic blood product that requires cross-matching and thawing prior to administration and is associated with immunologic reactions and possible pathogen transmission. Fibrinogen concentrate, a blood product alternative, is a purified form of fibrinogen, which undergoes a pasteurization process to minimize the risk of immunologic and allergic reactions. The primary aim of this study is compare the in vivo effect of post-CPB administration of FC, a blood product alternative, to cryoprecipitate on neonatal clot properties and clinical outcomes.

Arms:
arm 1: Neonates undergoing elective cardiac surgery requiring cardiopulmonary bypass (CPB) who are randomized to receive platelets and FC after separation from bypass. The dose of fibrinogen concentrate will be calculated to achieve a level of 300mg/dL after drug administration.

If a patient in either arm continues to have post-bypass bleeding, the anesthesiologist will use point of care testing and the transfusion thresholds outlines in the transfusion algorithm to determine appropriate products for transfusion. arm 2: Neonates undergoing elective cardiac surgery requiring cardiopulmonary bypass (CPB) who are randomized to receive platelets and cryoprecipitate. Standard transfusion algorithm includes two units of cryoprecipitate, which result in a median post-operative fibrinogen level of 286mg/dL (based on findings by Downey et al., published in Anesthesia and Analgesia in 2020).

If a patient in either arm continues to have post-bypass bleeding, the anesthesiologist will use point of care testing and the transfusion thresholds outlines in the transfusion algorithm to determine appropriate products for transfusion.

Interventions: intervention 1: Fibrinogen Concentrate (FC) intervention 2: Cryoprecipitate
Intervention descriptions: intervention 1: The dose of fibrinogen concentrate will be calculated to achieve a level of 300mg/dL after drug administration. intervention 2: The standard transfusion algorithm includes two units of cryoprecipitate, which result in a median post-operative fibrinogen level of 286mg/dL.

Protocol document:
SAP_002.pdf: 
Statistical Analysis Plan 
 
Title: In Vivo Effects of Fibrinogen Concentrate (FC) Versus 
Cryoprecipitate on the Neonatal Fibrin Network Structure After 
Cardiopulmonary Bypass (CPB) 
 
NCT Number: NCT03932240 
 
Date of Final Version: November 30, 2023 


In Vivo Effects of Fibrinogen Concentrate (FC) Versus Cryoprecipitate on the 
Neonatal Fibrin Network Structure After Cardiopulmonary Bypass (CPB) 
Statistical Analysis Plan 04.01.2019 (edited 11.30.23) 
Study Populations: 
Intention-to-Treat (ITT) Analysis Set 
The ITT Analysis Set will consist of all enrolled subjects according to their randomization 
assignment.  Efficacy summarization and analysis will be conducted using the ITT analysis set 
and this will the primary analysis.  
Per Protocol Analysis Set (PPAS): 
The subset of subjects in the full analysis (ITT) set who complied with the protocol sufficiently 
(see below) without any major protocol deviations. Confirmatory analysis will be performed 
using the PPAS dataset. Final definition of PPAS will be defined prior to data lock. Elements of 
PPAS may include exclusion criteria such as:  
• 
Violation of inclusion or exclusion criteria 
• 
Did not receive study medication  
• 
Did not go on cardiac bypass during surgical intervention 
• 
Incorrect study medication treatment  
• 
Patients unable to wean from bypass in operating room 
Sample Size Considerations 
The primary endpoint of the study, on which the statistical power is based, is the difference in 
clot degradation time at 24 hours post-operatively in neonates randomized to Cyro compared to 
those randomized to FC, with the primary focus on the overall set of subjects in the ITT 
population. Based on our previous work comparing adult and neonates, we observed a large 
difference (>2 standard deviation (SD) difference) in degradation times between the adults and 
neonates. In this study, we conservatively estimate a 1 SD difference in mean degradation 
times when comparing degradation times between infants randomized to Cyro vs. those 
randomized to FC. Assuming a two-sided test at the 5% significance level, 17 completed 
participants will provide 80% power to detect a 1 SD difference in mean degradation times 
between our two groups at 24-hours post-operative. Power was calculated using a two-sample 
t-test.  Additionally, assuming an attrition of 1 per group (i.e., death before 24 hour endpoint), 18 
participants are// needed for enrollment into the study to ensure an adequate number of 
subjects complete the primary endpoint. Power calculations were made using PASS v. 14.0.8 
(Kaysville, UT). 
General Statistical Considerations 
Descriptive statistics will be calculated for all variables of interest. For continuous variables this 
will include means and standard deviations, medians, quartiles (25th, 75th), minimum and 
maximum values. Categorical data will be described using counts and percentages. Continuous 
data will be assessed for normality using histograms and normal probability plots and 
statistically tested using the Anderson-Darling test and/or the Shapiro Wilk test for normality. 
Non-normal data will be presented using medians accompanied by 25th and 75th percentiles. 


No adjustments will be made for testing multiple secondary and exploratory efficacy outcomes. 
Given the large number of secondary/exploratory outcomes, the p-values associated with such 
outcomes will be considered descriptive as the likelihood of making a type I error is inflated due 
to multiple testing. All analyses will be conducted using SAS v. 9.4 (Cary, NC) or CRAN R v. 3.3 
or above (Vienna, Austria). All statistical testing will be two-sided and performed using a 0.05 
significance level (i.e., alpha =0.05). Missing values at baseline will be imputed using group 
median values at the specific time points. We will also repeat all analyses without imputation as 
a sensitivity analysis.  
Analysis of Primary and Secondary Outcomes: 
The primary endpoint of the study is the percentage change of normalized degradation at 24h 
post-surgery. Primary comparison will be the difference of the primary endpoint between 
neonates randomized to Cyro and those randomized to FC, with the primary focus on 24h post-
surgery time point. To answer this primary research question, we will utilize two sample t-test or 
Wilcoxon rank sum tests to compare percent change from pre-surgery (baseline) between 
groups at 24h post-surgery. We will further employ linear mixed models to analyze the 
longitudinal endpoints accounting for repeated measures. Specifically, we will examine 
percentage change of normalized degradation at follow up time points (i.e., post-surgery, ICU 
admission, and 24h post-surgery) relative to baseline between the two groups.  Random 
intercepts at patient level will be included to account for within-subject correlation. Post-hoc 
comparison between time points will be conducted by contrasts. Results will be presented as 
difference in least square means between groups or between time points, with associated 95% 
confidence intervals. Analysis of secondary outcomes, including measurements collected post-
transfusion, will be analyzed using similar methods.  
Finally, two sample t-tests or Wilcoxon rank sum tests will be used to examine the difference in 
clot kinetics (i.e., structure, polymerization, degradation) and clinical outcomes including chest 
tube output, 24-transfusion needs, and ICU length of stay. Two-sample t-tests or Wilcoxon rank 
sum tests will be used to compare measures between patients with and without thrombotic 
events. Multivariable linear regression for continuous outcomes and logistic regression for 
binary outcomes will be used to examine the association between the clot kinetics and clinical 
outcome while adjusting for potential confounders including pre-surgical weight, age, cardiac 
diagnosis/ RACHS score, and pre-operative comorbid conditions, as needed.  
 
Exploratory Subgroup Analysis 
Given the sample size of our current study, we are likely underpowered to examine differences 
in outcomes when stratified by subgroups including race, sex, risk of bleeding and underlying 
morphology. Nevertheless, we will perform subgroup analyses to examine differences in primary 
and secondary outcomes within and between subgroups of interest. This will include stratified 
analyses and by including the interaction between treatment group (FC vs. Cryo) and subgroup 
in subsequent models.  

Prot_001.pdf: 
Version 5/16/2019 
 
 
Title: In Vivo Effects of Fibrinogen Concentrate (FC) versus Cryoprecipitate on the 
Neonatal Fibrin Network Structure after Cardiopulmonary Bypass (CPB) 
 
Protocol version date: May 16, 2019 
 
NCT number: NCT03932240 
 
 
 


Version 5/16/2019 
 
SPA YIG Protocol 
 
Title: In Vivo Effects of Fibrinogen Concentrate (FC) versus Cryoprecipitate on the 
Neonatal Fibrin Network Structure after Cardiopulmonary Bypass (CPB) 
 
Principal Investigator: Laura A Downey, MD 
Co-PI: Nina Guzzetta, MD 
 
Abstract 
 
Like most organ systems in the newborn, the hematologic system is not fully developed 
at birth. Studies have shown both quantitative and qualitative deficiencies in many pro- 
and anticoagulation proteins (1). Neonates with congenital heart disease (CHD) often 
require cardiac surgery and cardiopulmonary bypass (CPB) within a few days of birth.  
Their immature coagulation system, effects of CPB, and the complexity of the surgical 
repair contribute to the potential for a substantial coagulopathy following CPB.  
Transfusion of red blood cells (RBCs), platelets, fresh frozen plasma (FFP), and 
cryoprecipitate is often needed to restore post-CPB hemostasis. However, excessive 
bleeding and perioperative transfusions are independent predictors for morbidity and 
mortality (2-4). Therefore, developing strategies to reduce bleeding and transfusion 
requirements in neonates after CPB is an urgent need. 
 
The most common hemostatic derangement after CPB is an acute acquired 
hypofibrinogenemia, which compromises fibrin clot generation and platelet 
aggregation, resulting in increased bleeding and need for allogenic blood product 
transfusions.  Currently, management of this acquired hypofibrinogenemia is replaced 
with a blood product, cryoprecipitate. However, fibrinogen concentrate, RiaSTAP (CSL 
Bering), is a commercially available product that is being used off-label in cardiac and 
pediatric surgery to reduce bleeding and transfusions.  Riastap, made from human 
plasma, contains purified fibrinogen as a pasteurized, lyophilized powder that has 
several advantages over cryoprecipitate: 1) small volume; 2) accurate dosing; 3) shorter 
preparation time since no blood typing or thawing are required; 4) lower risk of 
infectious transmission; 5) lower risk of allergic and immunologic reactions (5). In 
addition, recent studies have demonstrated that fibrinogen in neonates exists in a 
neonatal form that is distinct from the adult form of fibrinogen.  Of particular concern, 
clots formed from a mixture of adult and neonatal fibrinogen, simulating transfusion, 
took twice as long to degrade as clots containing pure neonatal fibrinogen.  These 
results raise concerns about the ability of the immature neonatal fibrinolytic system to 
degrade mixed clots and therefore a potential increased risk of adverse thrombotic 
events after the administration of adult fibrinogen.  However, a study specifically 
targeting neonates undergoing open-heart surgery with CPB has not been done to 
determine the efficacy of fibrinogen concentrate in this population.  
 


Version 5/16/2019 
 
In this study, we plan to complete a randomized, control trial in neonates undergoing 
cardiac surgery to characterize the in vivo effect of transfusion with either 
cryoprecipitate or fibrinogen concentrate on clot kinetics and degradation. Our 
secondary goal is to correlate individual clot properties to patient outcomes, specifically 
the degree of postoperative bleeding, transfusion requirements and signs or symptoms 
of a postoperative thrombosis.  
 
Introduction and Background 
 
In patients undergoing open-heart surgery (OHS), transfusion of allogenic blood 
products is independently associated with increased morbidity and mortality (6,7). In 
response to this, research in adult cardiac anesthesia has focused on blood product 
usage and developing algorithms to limit blood product exposure. Due to the smaller 
population, considerable variability in patient characteristics, and wide range of 
procedures performed in pediatric cardiac patients, the literature regarding blood 
conservation strategies for pediatric cardiac patients is limited. In children undergoing 
OHS, there are no published algorithms even though product exposure, particularly in 
neonates, is significant. The National Heart, Lung and Blood Institute (NHLBI) has 
determined pediatric transfusion medicine (PTM) to be a high priority for clinical and 
translational research, specifically targeting transfusion strategies that could reduce 
short and long-term morbidity and mortality (8).  
 
Congenital heart defects are the most common type of birth defect, affecting 
approximately 1% of births in the US (9).  Up to 20% of these patients require cardiac 
surgery within the first 30 days of life (9).  Neonates are extremely susceptible to the 
coagulopathic effects of CPB due to an immature coagulation system, massive 
hemodilution from large circuit primes, and extensive suture lines that accompany 
complex congenital cardiac repairs (10-11). While all neonates requiring CPB experience 
bleeding, up to 25% may suffer excessive bleeding requiring massive transfusions (10). 
Increasing transfusions of allogenic blood products during pediatric cardiac surgery are 
associated with increased mortality, prolonged mechanical ventilation, prolonged 
hospital stays, increased risk of infections, kidney injury, and negative immunologic 
responses (3-4-7). Given the safety concerns and the effects on the immune system 
associated with blood product administration, the search for alternative options to treat 
coagulopathy remains an important challenge.  Unfortunately, there exists a paucity of 
adequately studied pharmacologic options to reduce post-CPB bleeding in neonates. 
Therefore, the aim of this study is to evaluate an effective, yet safe, pharmacologic 
alternative to blood transfusions for managing post-operative bleeding in neonates after 
CPB.  
 
Like most organ systems in the newborn, the hematologic system is not fully 
developed at birth, but the parallel maturation of the coagulation and fibrinolytic 
systems maintains the hemostatic balance between thrombosis and bleeding. Several 


Version 5/16/2019 
 
studies have demonstrated that neonates have quantitative and qualitative deficiencies 
in many pro- and anticoagulation proteins (1, 15-17). Specifically, there is a “fetal 
fibrinogen” that is qualitatively dysfunctional and exists in a fetal form until 
approximately one year of age (15). In fact, several ex vivo studies have demonstrated 
improved fibrin network structure in neonatal pediatric cardiac patients (1) with the 
addition of adult fibrinogen.  However, Brown et al (1) demonstrated that adult 
fibrinogen does not integrate seamlessly with neonatal fibrinogen and may increase clot 
degradation times, thus increasing the risk of post-operative thrombosis.  
 
In addition to an immature hematologic system, fibrinogen, the final component 
of the clotting pathway, is one of the earliest coagulation proteins to become depleted 
during major bleeding and surgery with CPB.  Studies have reported worse outcomes 
in patients with lower fibrinogen levels in multiple clinical settings including trauma, 
cardiac, and pediatric surgeries (18-22).  As a result, an increasing body of literature has 
demonstrated that targeting higher fibrinogen levels reduced blood loss and 
transfusions in pediatric patients undergoing craniofacial and scoliosis surgery (19).  
The available options for fibrinogen replacement are cryoprecipitate and FC. In the 
United States, cryoprecipitate has been the traditional source of fibrinogen. 
Cryoprecipitate contains a high concentration of fibrinogen, around 15 g/L (23), and the 
procoagulant factors vWF, FXIII, and FVIII (23)).  Cryoprecipitate is prepared as single 
units, which are pooled prior to administration with a typical dose being approximately 
2-3 units for a neonate.  The risk of viral transmission is similar to that of FFP, the 
fibrinogen concentration is variable, and blood group matching is needed (23). The time 
to thaw cryoprecipitate is often 45-90 minutes. Cryoprecipitate was withdrawn from 
most European countries years ago due to safety concerns, but remains available in the 
USA. In contrast, FC contains purified fibrinogen without other coagulation factors.  
Viral inactivation during the manufacturing process minimizes the risk of viral 
transmission (5). The concentration of fibrinogen is standardized, and the 
administration volume is low (5).  In addition, the time to availability is short as it does 
not require cross-matching or thawing. FC has been effective and well tolerated in 
various clinical settings. As a result, FC is being used off-label for the treatment of 
bleeding in pediatric patients. Administration of FC to pediatric patients has proven 
successful in decreasing major blood loss in the setting of scoliosis and craniosynostosis 
surgery (19).  Galas et al. (20) demonstrated that FC was an effective substitute for 
cryoprecipitate in a post-bypass transfusion algorithm in older pediatric cardiac 
patients (ages: 1 month-5 years). However, to date, no study has examined the use of FC 
in neonates after cardiac surgery with CPB or how FC interfaces with the neonatal clot 
structure, kinetics, and degradation.  
 
This study represents a shift in the design of hemostatic therapy for neonatal 
coagulopathy. While many institutions continue to treat post-CPB bleeding in neonates 
with the transfusion of blood products consisting primarily of platelets and 
cryoprecipitate, Europe and an increasing number of pediatric institutions are replacing 


Version 5/16/2019 
 
cryoprecipitate with fibrinogen concentrate. Given the safety risks and immunologic 
effects associated with blood transfusions, the search for effective alternatives to blood 
products for managing post-bypass coagulopathy is essential. To date, the studies on 
neonatal clot structure and kinetics are based on ex vivo addition of adult fibrinogen.  
Therefore, this is the first in vivo study to directly compare the effect of cryoprecipitate 
or FC on the neonatal fibrin network after CPB surgery. Our first hypothesis is that clots 
formed from FC will display similar structural characteristics as those formed with 
cryoprecipitate.  However, because FC lacks FXIII, an important factor for forming a 
stable fibrin network but also causing greater resistance to degradation (14), we also 
hypothesize that clots formed from FC will be degraded faster by the neonatal 
fibrinolytic system, thus reducing the thrombotic risk associated with transfusion of 
adult blood products. This work is significant because it is the first in vivo study to 
specifically 1) compare in vivo transfusion of cryoprecipitate to FC on neonatal clot 
structure, strength, and kinetics, 2) assess the degradation rates of two types of adult 
fibrinogen by the neonatal system, and 3) correlate clot structure and degradation with 
clinical outcomes.  
 
Significance: 
 
Overall Aim: Determine the effects of post-CBP fibrinogen transfusion on the 
neonatal fibrin network to develop effective and safe strategies for managing 
coagulopathies in neonates. This proposal compares the in vivo effect of post-CPB 
administration of FC, a blood product alternative, to cryoprecipitate on neonatal clot 
properties. Our previous studies using cryoprecipitate suggest that adult fibrinogen 
does not seamlessly integrate with neonatal fibrinogen and may result in prolonged clot 
degradation, thus increasing the risk of thrombotic events. It is unclear if this is a direct 
result of the adult fibrinogen molecule or the other clot stabilizing factors in 
cryoprecipitate. This is the first in vivo study to 1) compare transfusion of 
cryoprecipitate to FC on neonatal clot structure, 2) assess the degradation rates between 
the two types of fibrinogen replacement, and 3) correlate clot structure and kinetics 
with clinical outcomes.  
 
Central Hypothesis: We hypothesize that FC clots will have similar structural 
characteristics to clots formed from transfused cryoprecipitate, but that FC clots will be 
degraded faster by the neonatal fibrinolytic system. 
 
Specific Aims: 
Aim 1A: Characterize coagulation factors and properties of clot structure, 
strength, and polymerization kinetics in neonates undergoing CPB surgery after the 
transfusion of either FC or cryoprecipitate.  
Hypothesis 1A: Clots formed after administration of FC will have similar strength 
and polymerization kinetics as clots formed with cryoprecipitate. 
 


Version 5/16/2019 
 
Aim 1B: Compare clot degradation rates in neonates undergoing CPB surgery 
prior to transfusion of adult fibrinogen, immediately after the administration of FC or 
cryoprecipitate, upon arrival to the ICU, and at 24hours post-operatively.   
Hypothesis 1B: Clots formed after the administration of FC will have faster 
degradation times at 24hours than those formed with cryoprecipitate.  
 
 
Aim 2: To complement aims 1A and 1B, we will examine the correlation of clot 
structure and kinetics to clinical outcomes with specific attention to: 1) transfusion 
requirements within 24 hours of surgery; 2) 24-hour chest tube output; 3) evidence of 
adverse thrombotic events. 
 
Hypothesis 2: Patients who have faster clot degradation times will have decreased 
risk of post-operative thrombotic events, but no difference in transfusion requirements 
or chest tube output.  
 
Summary: The proposed work represents an innovative and radical shift in managing 
neonatal coagulopathy. Neonatal CPB patients receive multiple blood products, 
consisting primarily of platelets and fibrinogen (cryoprecipitate) to reestablish 
hemostasis after CPB.  These procedures are often complicated by excessive bleeding 
and repeated transfusions, which are associated with increased mortality and 
morbidity. Blood products are prepared from adult blood, and our recent studies have 
demonstrated that components of the adult coagulation system, namely fibrinogen, do 
not integrate seamlessly with the neonatal coagulation system.  Therefore, developing 
alternative strategies to reduce blood transfusions and achieve hemostasis are critical. 
This study proposes to evaluate how two fibrinogen replacement therapies – FC and 
cryoprecipitate – interface with neonatal fibrin network properties.  We will enroll 36 
neonates undergoing elective cardiac surgery and randomize them to receive either 
cryoprecipitate or FC as part of the post-CPB transfusion protocol. We will collect blood 
at 4 time points 1) baseline; 2) post-transfusion of platelets and either cryoprecipitate or 
FC; 3) upon arrival to the ICU; 4) 24 hours after surgery.  The samples will be used to 
measure coagulation factors, analyze the fibrin network structure by confocal and 
scanning electron microscopy, strength parameters by rheology and atomic force 
microscopy (AFM), polymerization by absorbency and thrombin clotting times, and 
degradation kinetics by a customs fluidics device.   
 
Experimental Design and Methods: 
 
In this proposal, we aim to compare the effects of post-bypass transfusion of 
cryoprecipitate to fibrinogen concentrate (FC) on neonatal clot structure, clot kinetics, 
and clot degradation times. We will further characterize the levels of important 
procoagulants (thrombin, FXIII, vWF, fibrinogen) throughout the perioperative process 
to allow for a direct comparison between the current standard of care, cryoprecipitate, 
and an alternative source of fibrinogen, FC. Additionally, studies have demonstrated 
increased risk of thrombosis associated with more blood transfusions (24-25) or elevated 


Version 5/16/2019 
 
levels of vWF (25) in neonates and infants after CPB. As demonstrated by our group, ex 
vivo mixtures of purified neonatal fibrinogen and purified adult fibrinogen (derived 
from cryoprecipitate) alter clot structure and have prolonged degradation times, 
increasing the potential for post-transfusion thrombosis. However, these studies were 
carried out using clots formed in vitro from the addition of purified neonatal and adult 
fibrinogen, which do not fully mimic the structure of clots formed in vivo. Therefore, we 
plan to carry out a prospective, randomized control trial to examine the in vivo effects of 
cryoprecipitate versus fibrinogen concentrate as an alternative treatment for post-CPB 
coagulopathy in neonates undergoing cardiac surgery (Figure 6).   
 
Study Design: This study is a prospective, randomized control trial designed to evaluate 
fibrinogen concentrate as an alternative therapy to cryoprecipitate in treating post-CPB 
coagulopathy in neonates undergoing cardiac surgery.  After informed consent is 
obtained, we will randomize infants undergoing cardiac surgery to receive 
cryoprecipitate (standard of care) or fibrinogen concentrate (FC) (study arm) as part of a 
post-bypass transfusion algorithm. For patients enrolled in the study, we will follow 
our standard anesthetic management, cardiopulmonary bypass protocol, and 
transfusion thresholds in the operating room and ICU.  Patient demographics, relevant 
clinical data, standard laboratory data, and adverse events will be collected for each 
subject. In addition to our standard of care labs, whole blood samples will be collected 
from each study patient to analyze various properties of the neonatal fibrin network, 
including structure, strength, and polymerization and degradation kinetics, after the 
transfusion of either cryoprecipitate or FC.  These studies will allow us to directly 
compare clotting dynamics in the presence of the current standard of care for treating 
surgically induced coagulopathy, adult cryoprecipitate, and the potential alternative 
therapy FC.   
 
Study Population: As discussed above, neonates have unique aspects of their coagulation 
cascade that warrant specific biological consideration. This study will be a single-center 
prospective, randomized control trial.  After informed and written parental consent, a 
total of 36 neonates (age less than 30 days) undergoing elective cardiac surgery with 
cardiopulmonary bypass will be enrolled at Children’s Healthcare of Atlanta. The 
following criteria will be used to determine patient eligibility: 
      
Inclusion Criteria: 
 
1. Full term neonates (36-42 weeks gestational age)  
             2. Infants < 30 days of age at time of surgery 
             3. APGAR score of 6 or greater at 5 minutes after delivery 
      4. Neonates undergoing elective cardiac surgery requiring CPB at Children’s 
Healthcare of Atlanta 
      5. Parents willing to participate and able to understand and sign the provided 
informed consent 
 


Version 5/16/2019 
 
Exclusion Criteria: 
     1. Preterm neonates (less than 36 weeks gestation) 
     2. Patients undergoing an emergent procedure or surgery not requiring CPB 
     3. Patients with personal or family history of a coagulation defect or 
coagulopathy 
     4. Parents unwilling to participate or unable to understand and sign the provided 
informed consent 
 
Recruitment and Enrollment After a patient is deemed eligible for the study, informed 
and written consent will be obtained from the parents or legal guardian. Only those 
patients who meet the entry criteria will be approached for study participation.  There 
will be no costs for the participants.  At any time, parents and legal guardians will have 
the option of withdrawing their child from the study.  
 
Randomization As they present to Children’s Healthcare of Atlanta, patients will be 
enrolled consecutively and randomized to receive either FC or cryoprecipitate as part of 
a standardized transfusion algorithm.  Once on CPB, patients will be randomized using 
block randomization with variable blocks. Patients will be randomly assigned (1:1) to 
receive either cryoprecipitate (control group) or fibrinogen concentrate (FC) (study 
group) as part of our post-bypass transfusion algorithm. Ideally, all practitioners would 
be blinded to the study. However, the dose of fibrinogen concentrate is diluted in 
normal saline and may be up to approximately 50mls.  The use of a placebo would 
require the administration of up to 50mls of normal saline, 20% of a blood volume in a 
3kg neonate. The hemodiluational effects of placebo would potentially confound the 
results comparing the two groups. Therefore, the anesthesiologist, who will be 
administrating either cryoprecipitate or FC, will be aware of the group allocation.  
Parents, surgeons, and critical care physicians will be blinded to the group allocation. 
 
Rationale for Standardized Anesthetic, Cardiopulmonary Bypass and Anticoagulation 
Management In order to minimize confounding factors, we will follow our standard 
anesthetic management, cardiopulmonary bypass protocol, and anticoagulation 
strategy.  
 
Anesthesia Management and Cardiopulmonary Bypass Management 
Anesthesia, CPB, and surgery will be performed by the same teams of anesthesiologists, 
perfusionists, and surgeons.  Anesthesia techniques will follow institutional protocol, 
which include a balanced anesthesia technique and necessary invasive monitoring, 
including arterial and central pressure monitoring.  
 
Cardiopulmonary Bypass management 
Nonpulsatile CPB is performed using a nonheparin-coated system, a Terumo FX-05 
hollow-fiber membrane oxygenator (Terumo Cardiovascular Systems, Ann Arbor, MI), 
and Livanova SMArt-coated neonatal circuit (LIVANOVA USA (Sorin Group USA), 


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Arvada, CO, USA).  All circuits contain a 250-300 ml priming volume, which include 
50ml 25% albumin (Grifols), Plasmalyte, 0.5mg/kg mannitol, calcium gluconate, and 
8.4% sodium bicarbonate to normalize the pH. Packed RBCs (pRBCs) are added to the 
circuit as needed to achieve and maintain a hematocrit of 30% throughout the duration 
of CPB.  In accordance with institutional policy, patients having cardiac surgery receive 
pRBCs that are less than 14 days old. 50ml fresh frozen plasma (FFP) is added to CPB 
prime and another 50 ml FFP is added upon re-warming. Conventional or zero-balance 
ultrafiltration is performed throughout the bypass period at both institutions.  For all 
patients, pH-stat blood gas management is used during bypass.  
 
Anticoagulation 
Anticoagulation is established with 500units/kg of porcine heparin with 1000 units of 
heparin added to the CPB prime. Kaolin-activated ACT values greater than 480 seconds 
are confirmed prior to the initiation of CPB (i-STAT®1 Analyzer; Abbot Point of Care 
Inc, Abbott Park, IL, USA). Additional heparin is administered as necessary during CPB 
to maintain an ACT >480 seconds.  After completion of surgery, patients are rewarmed 
to 36oC and weaned from CPB. Protamine (5mg/kg) is used to neutralize heparin upon 
completion of CPB and confirmed with ACT. Additional protamine is administered if 
ACT levels are above baseline values. All neonates (patients <30 days of age) will 
receive anti-fibrinolytic therapy.  Tranexamic acid was used: 100mg/kg bolus dose pre-
bypass, 100mg/kg bypass dose, and an infusion at 10mg/kg/hr that was terminated at 
transport to the ICU. 
 
Transfusion Algorithm: In order to minimize confounding factors, a transfusion 
algorithm and transfusion thresholds will be followed in the operating room and ICU 
for all patients enrolled in the study.  RBCs will be transfused for hematocrit <32% for 
acyanotic neonates, while cyanotic neonates are transfused RBCs for a goal Hct 35-40%.  
RBC transfusion is not permitted for hematocrit values higher than 45%.   
 
After enrollment in our study, patients will be randomized to receive either 
cryoprecipitate or fibrinogen concentrate as part of our transfusion protocol.  Our 
standard transfusion algorithm for neonates includes a quarter to half a unit of platelets 
and two units of cryoprecipitate, which historically resulted in a median post-operative 
fibrinogen level of 345mg/dL (258-469) (unpublished data).  Using recent studies as 
well as our historical data, we chose a post-transfusion target fibrinogen level of 
300mg/dL.  After separation from bypass, patients will receive platelets and either 
cryoprecipitate or FC. For patients randomized to the FC arm, we will calculate the dose 
of fibrinogen concentrate to achieve a level of 300mg/dL after drug administration 
using the following equation from Riastap ® Package insert (5):  
 
 
 
 


Version 5/16/2019 
 
 
 
Dose = (Target Level-Measured Level) 
                            
 1.7 x weight(kg) 
 
 
 
In order to account for the hemodilutional effects of the bypass prime on fibrinogen 
levels, the “measured level” will be the fibrinogen level measured after 10min on CPB. 
If a patient in either arm continues to have post-bypass bleeding, the anesthesiologist 
will use point of care testing and the transfusion thresholds outlines in the transfusion 
algorithm to determine appropriate products for transfusion.   
 
Sample Procurement and Processing– At Children’s Healthcare of Atlanta, whole blood 
samples will be collected from 36 neonates scheduled for elective cardiac surgery 
requiring CPB. We will collect these samples at four time points: 1) after induction of 
anesthesia prior to CPB; 2) after termination of CPB and transfusion of platelets and 
either cryoprecipitate or fibrinogen concentrate; 3) upon arrival to the ICU 4) 24 hours 
post-operatively in the ICU.  These samples will be centrifuged to yield platelet poor 
plasma (PPP) and stored at -80oC. These samples will be utilized for the clot analysis 
experiments described in detail below.  Samples will be transported according to 
appropriate regulations to the laboratory of Dr. Brown at North Carolina State 
University for analysis. All samples will be obtained from an arterial line that is 
required for the planned surgical procedure. 
 
Analysis of coagulation factors – For each sample, we will measure coagulation factor 
levels (thrombin, FXIII, vWF, and fibrinogen) using ELISA to elucidate the contribution 
of these factors present in cryoprecipitate to clot kinetics when compared to FC. Each 
sample will be analyzed in triplicate to ensure that reproducible and statistically 
significant data are obtained. 
 
Analysis of clot characteristics - Clots formed from pre-CPB PPP, post-CPB PPP after 
transfusion of cryoprecipitate or FC, and 24 hours post-operative PPP will be analyzed 
to compare the effects of FC and cryoprecipitate on clot properties. Using previously 
described methods, strength parameters will be assessed by rheology and atomic force 
microscopy (AFM) and polymerization will be determined by thrombin-initiated 
turbidity/absorbency curves (13).  These studies will allow us to directly compare 
differences between fiber alignment (3-D branching structure), strength, and 
polymerization differences after the transfusion of FC or cryoprecipitate in neonates 
after CPB surgery. 
 
Analysis of fibrin fiber alignment – We will quantify clot fiber alignment using an 
application of an algorithm based on a fast Fourier transform (FFT) that our group has 


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previously employed (25). Briefly, each confocal image is padded with redundant data; 
a Gaussian decay (SD=0.25) and tow-dimensional Hann window function are applied to 
minimize edge effects; and the images are transformed into the frequency domain using 
a two-dimensional fast Fourier transform. The predominant fiber angle is defined as the 
angle with the maximum relative intensity, and the alignment index (AI) is defined the 
fraction of fibers aligned within +200 of the preferred fiber alignment (PFA) normalized 
to a random distribution of oriented fibers (1). The higher the AI, the greater alignment 
of the fibers.  Our previous work demonstrated that clots formed from purified 
neonatal fibrinogen were found to display a significantly higher degree of alignment 
(i.e. less 3-D structure) compared to those comprised of adult fibrinogen. Although we 
have not completed in vitro studies to analyze the effect of FC on neonatal clot 
formation, we expect that it will be similar to fibrin alignment in clots formed from 
purified adult fibrinogen derived from cryoprecipitate.  In support of our hypothesis, 
we expect that both FC and cryoprecipitate will have lower AI than baseline neonatal 
only clots, but there will likely be no difference between clots formed with FC or 
cryoprecipitate. Any difference in AI between the two groups will shed light on how 
pro-coagulant factors present in cryoprecipitate may affect in vivo clot formation in 
neonates after surgery with CPB (13) 
 
Analysis of clot degradation kinetics – To evaluate clot degradation kinetics, we will utilize 
a previously described custom fluidics-based microscopy approach (1).  Our previous 
data have demonstrated that clots from mixed neonatal and adult fibrinogen degrade 
slower than neonatal fibrinogen only clots.  Given that cryoprecipitate contains both 
vWF, (essential to the proper functioning of neonatal platelets) and FXIII (important for 
stabilizing clot strength), it may be that transfusion of adult-derived cryoprecipitate 
generates a clot that is not as easily degraded by the neonatal fibrinolytic system when 
compared to FC, which contains fibrinogen only.  FC may have faster degradation times 
when compared with cryoprecipitate, thus potentially reducing post-transfusion 
thrombotic risk in neonates after cardiac surgery with CPB (1).   
 
Aim 1A (Clot structure, strength, polymerization kinetics between 
cryoprecipitate and FC) Thirty-six neonates undergoing non-emergent cardiac surgery 
will be enrolled in this study and randomized to receive either cryoprecipitate or FC as 
part of post-bypass transfusion algorithm.  Blood samples will be collected at four times 
points: 1) after the induction of anesthesia 2) after transfusion of cryoprecipitate or FC; 
3) upon arrival to the ICU; 4) 24 hours after surgery.  After samples are collected and 
processed, they will be transported to Dr. Ashley Brown’s lab at NCSU/UNC-CH.  Dr. 
Brown will use ELISA to measure the coagulation factors (thrombin, FXIII, von 
Willebrand Factor, and fibrinogen) at each time point.  Using the previously described 
methods, strength parameters will be assessed by rheology and atomic force 
microscopy (AFM); polymerization will be determined by thrombin-initiated 
turbidity/absorbency curves; fiber alignment using the described fast Fourier 
transform. Our goal is to characterize how these two sources of adult fibrinogen affect 


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properties of the neonatal fibrin network including the structure, strength, 
polymerization kinetics between these two groups.  
 
Aim 1B (Clot degradation kinetics) In addition to the above studies, the blood 
samples from the same 36 neonates will then be used to perform degradation kinetic 
studies as described previously.  This will be the first study detailing the longitudinal 
differences in degradation kinetics of neonates undergoing cardiac surgery.  After 
characterizing the coagulation factor levels between patients who receive 
cryoprecipitate versus FC, we hope to better understand the role of vWF and FXIII in 
neonatal clot formation and degradation rates and how this may contribute to the risk 
of thrombotic events.  
    
Aim 2 (Correlation of clot properties to clinical outcomes): Clinical outcome 
variables will be collected for each patient and correlated to clot properties with specific 
attention to: 1) transfusions within the first 24 hours of surgery, 2) 24-hour chest tube 
output, 3) clinical signs and symptoms of postoperative thrombosis. Our multi-site 
prospective, randomized control trial demonstrated that FC can reduce allogenic blood 
transfusion in neonates and infants undergoing elective cardiac surgery with CPB when 
compared to cryoprecipitate. Our proposed laboratory studies use platelet poor plasma 
to enable us to focus on the contribution of supplemental fibrinogen to neonatal clot 
kinetics.  However, since platelets, particularly the increased dependence on vWF in 
neonates, also play an important role in post-bypass hemostasis, we plan to correlate 
clinical characteristics and outcomes with our findings regarding clot kinetics. Clinical 
outcome variables will be collected for each patient and correlated to clot properties 
with specific attention to 1) transfusion requirements intraoperatively; 2) transfusion 
requirements within the first 24 hours of surgery; 3) post-operative bleeding recorded 
by 24 hour chest tube output; 4) clinical signs and symptoms indicative of postoperative 
thrombosis; 5) mechanical ventilation time; 6) length of ICU and hospital stay 7) 
adverse events within seven days of surgery.  While our study is not powered to for this 
outcome, we hope that these data will provide some insight in to the clinical differences 
between different sources of adult fibrinogen. 
 
 
Samples Size/Statistical Analysis 
 
Data collected will include demographic data, CBP data, transfusion data, laboratory 
data and clinical outcomes listed above. As we demonstrated in prior work, neonatal 
clots prior to transfusion displayed a significantly higher degree of alignment when 
compared to those composed of adult only or mixed neonatal and adult fibrinogen. 
Based on our previous data, to detect a difference in degradation times between clots 
formed with neonatal fibrinogen and clots formed with adult fibrinogen with 80% 
power, we will enroll at total of 36 patients, with 18 in patients in each arm. All 
quantitative data will be analyzed using a paired t-test or Wilcoxon rank-sum test to 


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determine differences between the two groups. Correlation will be determined by either 
Pearson correlation or Spearman rank correlation. Significance will be defined by a p-
value less than or equal to 0.05. 
 
 
Adverse Events Reporting 
 
Monitoring of Adverse Events (AE) and Serious Adverse Events (SAE) is an 
important aspect of clinical trials.  After enrollment, the research team will collect the 
adverse events from the medical record. SAEs will be reported to the IRB within the 
guidelines set by the IRB.  Any serious AE will be immediately reviewed by the PI and 
a senior cardiac anesthesiologist not involved in the study.  Quarterly, study subjects 
will be reviewed on a case-by-case basis by our Quality Improvement Officer and an 
experienced cardiac anesthesiologist not involved in the study for review of potential 
adverse events and whether or not the event was related to the study.  
Drug reactions including fever, chills, headache, allergic reactions, or thrombosis 
are potential risks to this study.  The most common adverse reactions observed in 
clinical studies (frequency >1%) with the study drug, fibrinogen concentrate, were fever 
and headache. The most serious adverse reactions observed are thrombotic episodes 
(pulmonary embolism, myocardial infarction, deep vein thrombosis) and anaphylactic 
reactions. These reactions are similar to those of blood transfusions and specifically, 
cryoprecipitate. The risks are also comparable to cryoprecipitate. Fibrinogen concentrate 
has been used in Europe and as part of transfusion algorithms around the United States, 
including our institution with no increased serious adverse events reported.  In our 
preliminary study, we received an IND exemption for the use of fibrinogen concentrate.  
Thirty patients under one year of age received fibrinogen concentrate instead of 
cryoprecipitate to treat postoperative bleeding. There was no significant difference in 
the rate of adverse events, including postoperative thrombosis, between the fibrinogen 
concentrate versus the cryoprecipitate group. Based on this data and the need to 
minimize transfusions in the neonatal population undergoing cardiac surgery with 
CPB, we are seeking an IND exemption for use of this medication in a randomized, 
control trial.  
Patient confidentiality is at risk since protected healthy information is included 
in the study data. This risk will be minimized by only recording information absolutely 
necessary to fulfill the study’s objectives. Information directly identifying patients will 
be excluded.  Once the samples are collected, a unique study number will be assigned to 
each sample. This unique number will be used to identify the patient in the research 
database. The database, along with the code linking the study patient to an assigned 
number, will be kept in a locked office.  
 
Data Safety Monitoring Plan 


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A potential risk of this study is related to drug reactions including fever, chills, 
headache, allergic reactions, or thrombosis.  This medication has been used extensively 
in Europe and as part of transfusion algorithms around the United States.  Based on this 
data and the need to minimized transfusions in the infant population undergoing 
cardiac surgery with CPB, we have received an IND exemption for use of this 
medication in this randomized, control trial.  
 
The study subjects will be reviewed on a case-by-case basis at our institution and 
quarterly by the DSMB. The DSMB will consist of a cardiologist, an anesthesiologist, a 
hematologist, and a statistician not involved in the study. Serious adverse events, 
including thrombosis, stroke, sepsis, and death, will be reported to the DSMB within 24 
hours of the study team becoming aware of the event. The DSMB will review all safety 
data, including frequency and severity of AEs.  
 
A second risk of this study is patient confidentiality since protected health information 
is included in the study data. This risk will be minimized by only recording information 
absolutely necessary to fulfill the study’s objectives. Information directly identifying 
patients will be excluded (names, addresses, telephone numbers, social security 
numbers, email addresses, and account numbers). A unique study number will identify 
the study subjects. The study information will be collected and stored in a password-
protected database. Consents, along with the code linking a subject’s identity to an 
assigned number, will be locked in the office of the principal investigator or designee.  
 
The study team will conduct a self-monitoring tool quarterly to ensure data integrity.  
 
 
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