A Pilot Study Involving the Changes in the Markers of Coagulation During Cardiopulmonary Bypass Surgery Utilizing Acute Normovolemic Hemodilution
Conditions: Bleeding Reduction
Sponsor: Inova Health Care Services

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A Pilot Study Involving the Changes in the Markers of Coagulation during 
Cardiopulmonary Bypass Surgery Utilizing Acute Normovolemic Hemodilution 
 
 
 
Principal Investigator: Eric L. Sarin MD 
 
 
Sub-Investigator(s): Paul A. Gurbel MD, FACC 
 
 
Sponsor 
 
 
Site of Investigation: Inova Heart and Vascular Institute at Inova Fairfax Hospital,  
Falls Church, VA 
 
 
 
Date of Protocol:  12 April 2019 
 
 
 
 
Contact Information:  
Eric L. Sarin MD 
Inova Center for Thrombosis Research and Drug Development 
Inova Heart and Vascular Institute 
3300 Gallows Road 
Falls Church, VA 22042 
Phone: 703-280-5858 
Fax: 703-849-0874 
Email: eric.sarin@inova.org 
 
 
 
 
 
 
 
 
 
 

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PROTOCOL SYNOPSIS 
  
Title:   A Pilot Study Involving the Changes in the Markers of Coagulation during 
 
Cardiopulmonary Bypass Surgery Utilizing Acute Normovolemic Hemodilution  
 
 
Short Title:  TARGET ANH 
 
Rationale:  The results of this study will help enhance current understanding of the impact of 
Acute Normovolemic Hemodilution (ANH) administration on transfusion requirements post 
cardiac surgery, the effect of this procedure on coagulation, and platelet hemostasis, and the 
amount of chest tube drainage 24 hours post coronary artery bypass graft surgery (CABG) 
surgery. 
 
Objectives:   
Primary:  
The primary objective will be assessment of hemostasis at baseline and serially during 
coronary artery bypass graft surgery utilizing the cardiopulmonary bypass machine in patient 
utilizing acute normovolemic hemodilution. 
 
Study Type:  Single center study, prospective, observational, open label.  
 
Study Design:  A total of  60 subjects will be included in the study, 40 subjects, between the ages of 
18- 85 years old that will undergo first- time isolated (CABG) utilizing cardiopulmonary bypass 
(CPB) with the administration of ANH, and 20 controls undergoing first- time isolated CABG 
between the ages of 18- 85 years old not receiving ANH .  
 
Study Methodology:  We will analyze the hemostatic markers of 60 patients undergoing cardiac 
surgery utilizing cardiopulmonary bypass (CPB) pre and post ANH infusion, and without ANH 
infusion at various time points which shall be different for both the experimental (ANH recipients) 
and the control group (non-ANH recipients) culminating at 24 hours post CABG surgery to evaluate 
the need for allogenic blood transfusion, changes in coagulation and platelet hemostatic markers, 
and effect on 24 hour chest tube drainage. Time points for the control group (non-ANH recipients)  
will include seven blood draws (7): baseline, 30 minutes post-CPB machine initiation, once 
immediately off of CPB, and post protamine infusion. Further draws will be obtained at 1 hour 
post CPB withdraw, 2-4 hours post CPB withdraw, and finally at 16-24 hours post CPB 
withdraw. 
Time points for the experimental group (ANH recipients) will include eight (8) blood draws: 
baseline, 30 minutes post-CPB machine initiation, once immediately off of CPB, and post 
protamine infusion and after ANH reinfusion. Further draws will be obtained at 1 hour post ANH 
reinfusion, 2-4 hours post ANH reinfusion, and finally at 16-24 hours post ANH reinfusion.  
Statistical Methodology: Categorical variables will be expressed as n (%). Continuous variables 
will be reported as mean± (SD)], or median (interquartile range), where appropriate. Paired t-
tests or Wilcoxon signed rank tests or will be performed to test differences in lab values before 
and after treatment. Correlation analysis will be performed by Pearson’s or Spearman’s Rho, 
where appropriate. Univariate and multivariate logistic regression models will be used to 
determine predictors of clinical endpoints (24-hours chest output and total number of transfused 

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packed blood cells). The predictive cut-point values of platelet function tests to clinical endpoints 
will be assessed by receiver operator characteristic (ROC) curve analysis with Area Under Curve 
(AUC) statistics. We will use separate repeated measures generalized linear mixed models to 
associate changes in hemostatic markers, reduced chest-tube drainage, reduction in transfusion, 
etc. Statistical calculations will be carried out using SAS (Ver. 9.3, Cary, NC).  
 
1. INTRODUCTION 
 
1.1 Specific Aims 
 To better understand the relationship between ANH utilization during CABG and its 
effect on the quantity and character of specific biomarkers of thrombogenicity at 
baseline and serially pre- withdraw and post- reinfusion of the patients collected 
blood.  
 To identify variations in thrombogenic phenotypes which play a role in the 
decreased incidence of bleeding complications among patients undergoing ANH 
transfusion post CABG and those patients who do not undergo ANH transfusion. 
And to obtain pilot data to clarify the influence of the ANH procedure on clinical 
outcomes as evidenced by the incidence of perioperative allogenic blood transfusion 
and quantification of 24 hour chest tube drainage post CABG. 
 
1.2 Hypothesis  
We hypothesize that ANH will be associated with favorable changes in hemostatic markers, a 
reduced total number of transfused, packed red blood cells, and less chest tube drainage at 24 
hours after CABG surgery. We will also gain a better understanding the characteristics of the 
blood components intraoperatively and postoperatively which are responsible for these favorable 
outcomes. 
 
1.3 Background and Significance  
Cardiac surgery is foremost among procedures that consume allogenic blood transfusions. 
Excessive bleeding in cardiac surgery is multifactorial in that it can be caused by the surgical 
procedure itself or the effects of the surgery on hemostasis 1. And while allogenic blood transfusion 
has been identified as a major mechanism of harm in cardiac surgery patients it is balanced by the 
harmful sequelae of bleeding 2. Predictors for the need of allogenic blood transfusion include; 
advanced age, preoperative anemia, small body size, female sex, preoperative antithrombotic 
therapy, coagulation disorders, reoperation, and prolonged CPB time 3. Preoperative hematocrit is 
the most frequent indicator of intraoperative transfusion in CABG patients 4. The incidence of chest 
tube drainage greater than 1000 ml in the 24 hours post CABG surgery is a strong predictor of 
mortality in this patient population 2.  
 
The profound risk factors which exist for allogenic blood transfusion in the context of  CABG 
utilizing CPB are; infection, circulatory overload secondary to blood volume increase, immune 
suppression, transfusion mediated acute lung injury, hospital acquired infection, renal failure, 
stroke, febrile reactions, hemolytic reactions, severe sepsis, sternal wound infection, post- operative 
pneumonia, multiple organ failure, frequent admission to intensive care, increased length of hospital 
stay , hospital mortality, and most significantly an increased short and long term mortality 

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4,5,6,7,8,9,10,24. Patients undergoing CABG are also at risk for anemia related to perioperative bleeding, 
coagulopathy, hemodilution secondary to pump priming solutions 11, 12, 13 and pump trauma.  
The use of allogenic transfusion to increase tissue oxygenation in perioperative anemic patients 
while theoretically effective is disputed by many studies which demonstrate no increase in tissue 
oxygenation attributable to allogenic blood transfusion 14, 15. Hemodilution is a strategy that may 
avoid or limit the utilization of allogenic blood 16. 
Acute Normovolemic Hemodilution (ANH) involves the removal of the patients own blood 
immediately at the induction of anesthesia and replacement of that blood with an equivalent 
volume of crystalloid or colloid solution in order to dissuade the loss of RBC 17 cell mass, the 
mechanism of action is dilution of the blood, hematocrit reduction, and reduction of the 
anticoagulant components of the blood 18 associated with bleeding during CABG utilizing CPB. 
The removed blood is isolated from the negative effects of CPB such as the activation and 
consumption of platelets 19, the retention of RBC’s in the pump mechanism, and the destruction 
of blood products secondary to pump trauma. The collected blood is then stored in anticoagulant 
treated blood bags and reinfused into the patient in reverse harvest order 16 at the commencement 
of surgery. This reinfusion method ensures the highest quality blood, that richest in cell mass is 
administered last 25. Success of this blood product conservation technique is dependent upon the 
preoperative high hematocrit count, and volume of blood collected balanced with the amount of 
intraoperative blood lost 16, 17. 
A review of relevant literature identifies many situations in which ANH is beneficial; in patients 
that refuse blood transfusion for religious reasons ANH provides RBC’s, plasma, and platelets, 
in patients who have a preoperative high hematocrit count 20 ANH eliminates the risk of 
transfusion reaction inherent with allogenic blood components, preserves erythrocytes from CPB 
damage, enhances coagulation through the readministration of the patients own clotting factors 
and platelets, and improves perfusion via reduction of blood viscosity which in turn delivers 
more oxygen to the tissues by increasing blood flow to the stenosed and collateral circulation in 
the myocardium 21. ANH reverses the effects of CPB administered heparin and the reduced blood 
oxygen carrying capacity secondary to hemodilution 21.  ANH is easy to perform, low in cost, 
with no evidence of the untoward effects of coagulation, hemolysis, fibrinolysis, or negative 
immune response in the collected blood as opposed to RBC transfusion 21.  
However, the beneficial attributes of ANH are countered by studies that highlight; the time 
consuming nature of harvesting and reinfusing the patient’s blood, an initial low hemoglobin 
count limits the amount of blood that can be collected, and the role of intraoperative blood 
conservation has been touted to also reduce the need for allogenic transfusion without the 
disadvantages of ANH 22 collection and reinfusion.  Also confounding the benefits of ANH is the 
marked disparateness between trials of ANH as a blood conservation strategy, studies with 

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inadequate power, and frequency of studies not specifically designed to evaluate the efficacy of 
ANH and therefore devoid of definitive assessment of clinical benefit 17. 
In order to avoid the negative sequelae associated with allogenic blood transfusion, utilization of 
the blood conservation method of ANH will be an effective strategy for avoiding loss of cell 
mass during on pump CABG surgery. Exactly how this procedure is beneficial, most 
significantly its effects on platelets and clotting mechanisms is a significant gap in knowledge. 
We propose to study the effect in patients undergoing ANH on; the need for allogenic blood 
transfusion, hemostatic markers, and chest tube drainage 24 hours post CABG. Positive effects in 
reduced number of transfused RBC’s, the hemostatic markers of anticoagulation and platelets, 
and reduced chest tube drainage at 24 hours post CABG will indicate a positive correlation with 
ANH utilization during isolated on pump CABG surgery. We propose that this study will 
provide us with a better understanding of the effects of ANH on coagulation, specifically 
platelets and how ANH contributes to better outcomes associated with clotting. 
 
1.4 Preliminary Studies 
In our earlier analysis of 1,319 non- isolated CABG patients, we compared post- operative 
morbidity and mortality in patients with and without ANH. We found that ANH was associated 
with a reduced risk for intraoperative blood product use (OR= 0.41, p<0.001), including use of 
RBC (OR= 0.32, p<0.001) and fresh frozen plasma (OR=0.49, p<0.001). Transfusion of 
intraoperative RBC and FFP was independently associated with greater risk for prolonged 
mechanical ventilation (p<0.001 and p=0.003, respectively) and operative mortality (p=0.005 
and p=0.04). However, how ANH influences hemostasis in patients undergoing cardiac surgery 
is unknown 27. 
 
 
2. STUDY DESIGN AND SUBJECT SELECTION  
 
2.1 Study Type 
This is an observational study that will be conducted in subjects undergoing first- time isolated 
CABG surgery.  
 
 
2.2 Setting/Location 
Inova Heart and Vascular Institute, 3300 Gallows Road, Falls Church, VA 22042 
 
2.3 Duration of Study 
Patient participation will entail perioperative phlebotomy during the course of CABG surgery      
utilizing CPB and postoperatively with the last blood draw completed 16-24 hours after the 
cessation of CPB. Medical records will be reviewed for adverse events and discharge date upon 
hospital discharge. 
 
2.4 Number of Subjects  
Sixty (60) patients are expected to participate in this trial. 
2.5 Study Population 

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2.5.1 Population Characteristics 
       2.5.1.1 Gender and racial and ethnic origin of subjects 
       The study’s intended population is inclusive of both genders (males and females),                   
and all racial and ethnic groups and subgroups.   
 
2.5.1.2 Age of subjects 
        Subject enrollment will be comprised of subjects 18 to 85 years of age. 
 
2.5.2 Vulnerable Population 
               Children, pregnant women, institutionalized persons, and persons with decisional  
               incapacity will be not be enrolled in this study.  
 
2.6 Recruitment  
Recruitment will occur in one of two settings; while inpatient at the IHVI upon first contact with 
the cardiac surgery service, while hospitalized but prior to the day of surgery in order to give the 
patient and family time to review the consent, or outpatient at the Inova Cardiac Surgery offices 
at 2921 Telestar Court, Falls Church, Virginia. The patient will be considered enrolled in the 
study after signing informed consent. The expected length of the recruitment period is 8 months. 
Approximately 300 coronary artery bypass graft surgeries are performed at the IHVI each year. 
If the study conduct (e.g. recruitment rate; drop-out rate; data quality; protocol compliance) does 
not suggest a proper completion of the trial within the reasonable time frame as agreed upon, the 
recruitment period may be extended to reach the desired sample size. 
 
2.7 Inclusion Criteria 
 Subject is 18 to 85 years old. 
 Subject undergoing an isolated CABG procedure with or without ANH 
 Subject is hemodynamically stable 
 The subject is able to read and has signed and dated the informed consent document    
including authorization permitting release of personal health information approved     
by the investigator's Institutional Review Board (IRB). 
 
 
2.8 Exclusion Criteria: Subjects will be excluded from entry if ANY of the criteria listed below 
are met: 
 Prior CABG 
 Hematocrit <30 at baseline (ANH Group only). Hematocrit will be assessed by 
anesthesiologist and surgeon to determine if patient is eligible for control group 
 
 Insufficient (Low) on pump hematocrit of <21% 
 Patient is hemodynamically unstable 
 Subject requiring an emergency procedure 
 Left main coronary artery stenosis with evidence of hemodynamic instability (e.g. 
 hypotension, ST segment elevations on electrocardiogram). 
 Aortic valve stenosis (e.g. hypotension, ST segment elevations on 
 electrocardiogram). 
 
 
 

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3. STUDY METHODS AND PROCEDURES 
3.1 Study Procedures 
This trial involves serial blood sample collection starting prior to anesthesia administration until 
16 to 24 hours post- surgery A total of 60- 80  mLs of blood will be collected for research 
purposes throughout the study duration based on group designation (ANH recipients versus 
control). See timing of assessments table for specific laboratory tests drawn at each time pointfor 
each group.  
 
The screening process will consist of the routine pre-operative screening process which is 
standard of care for cardiopulmonary bypass surgery: imaging, laboratory blood draws and a 
complete history and physical examination. Those patients who meet all of the inclusion criteria 
and do not meet any of the exclusion criteria will be offered enrollment. 
 
Baseline shall be defined as the commencement of CABG surgery upon the induction of 
anesthesia, but before utilization of the coronary artery bypass machine.  Baseline laboratory 
assessments which will entail collection of the markers of coagulation and platelet function at the 
commencement of surgery upon induction of anesthesia and the insertion of the central venous 
catheter. During CPB just after the induction of anesthesia, a central venous catheter and other 
monitoring catheters are inserted into the patient. The insertions of these monitoring catheters are 
standard of care during CABG surgery. Prior to the start of the blood removal phase of the ANH 
procedure, blood samples will be obtained for baseline complete blood count, coagulation, 
inflammation, and hemostatic markers. A calculated amount of blood will then be removed and 
sequestered at room temperature in the cardiovascular operating room (CVOR) to protect it from 
the damaging effects of both the CPB machine and the procedure.  
 
Once the CPB machine has been initiated, the subjects on- pump hematocrit (HCT) will be 
calculated. A safety threshold for blood removal in the ANH process will be calculated 
according to the following formula by the perfusionist: 
 
EBV= fbv x kg  
 
 
 
 
 
Eq.1 
 
Hct pre-cpb= Hct on cpb x (EBV + Prime volume)  
Eq.2 
 
 
 
 
EBV 
 
ANH Volume= EBV x (Hct baseline – Hct pre-cpb)  
Eq.3  
 
 
 
 
Hct baseline 
 
 
 EBV= patient’s blood volume in milliliters. 
 Fbv= blood volume factor in cc blood per kilogram: usually 65-70ml 
 Kg = patient’s weight in kilograms 
 Hct pre-cpb = percent estimated patient Hct required before CPB to achieve the  desired 
Hct on cpb 

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 Hct on cpb = desired percent Hct after initiating CPB 
 Prime volume = extracorporeal circuit prime volume after retrograde autologous 
 
priming (if applicable). 
 Hct baseline = patient’s percent Hct after hemodilution after the induction of the 
 
anesthetic agent 
 ANH volume = whole blood volume in cc to be sequestered prior to CPB 
 
This assessment will determine whether subjects undergo ANH or do not have sufficient 
hematocrit to undergo ANH. Once determination of adequate on- pump hematocrit has been 
confirmed blood samples from the subject  will be collected 30 minutes post-CPB machine 
initiation, once immediately off of CPB, and post protamine infusion and after ANH reinfusion. 
Further draws will be obtained at 1 hour post reinfusion, 2-4 hours post reinfusion, and finally at 
16-24 hours post reinfusion in the experimental group  In the control group blood samples from 
the subject  will be collected 30 minutes post-CPB machine initiation, once immediately off of 
CPB, and post protamine infusion. Further draws will be obtained at 1 hour post CPB 
completion, 2-4 hours post CPB completion, and finally at 16-24 hours post CPB completion. 
While many of these blood draws are standard of care, others are trial-related (i.e. coagulation, 
inflammation, and hemostasis markers). 
 
The blood tests that are done at 1 and 2 hours post reinfusion are expected to be done in the 
CVOR or in the Cardiovascular Intensive Care Unit (CVICU) depending on the patient’s 
location. These samples will be obtained through the central venous catheter. The 16-24 hour 
blood draw will be done in the (CVICU) through the central venous catheter or phlebotomy 
depending on the time of discontinuation of the central line, and this will conclude blood 
sampling collection for the trial. 
 
Should the subject become hemodynamically unstable (systolic BP less than 90 mmHg) at any 
time within either the experimental or the control group  assessments will be stopped and 
standard of care will be continued as appropriate for the subject’s condition. Patient will continue 
to be followed for 30 days or as their condition dictates as per standard of care. 
 
Upon hospital discharge, medical records will be reviewed to record discharge date, assess for 
adverse events as specified in section 7.1, and to document the total number of allogenic blood 
transfusions.  
 
Should the subject consent to the use of remaining specimens, plasma samples for possible future 
analysis will be stored at minus 70- 80 degrees Celsius for up to 2 years.     
 
 
3.2 Endpoints/Outcomes Measurements 
 3.2.1 Primary Objective 
 The primary endpoint will be assessment of hemostasis at baseline and serially 
during CABG surgery utilizing the CPB. 
 
3.2.4     Informed Consent  

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The research staff will meet with the participant to discuss the goals of the study, possible  
risks of participation, and to answer any related to the procedure or to the study. The 
research staff will allow adequate time and privacy for decision making and then obtain a 
signed consent document. The subject will receive a copy of the signed consent form. 
Patients incapable of providing consent due to a medical situation will not be approached for 
study participation. No study related procedures may commence without a signed consent 
form. A separate signature will be required to document a subjects agreement to allow any 
remaining specimens obtained during the course of participation to be used for ancillary 
research. Subjects who decline to participate in this optional research will not provide this 
separate signature and any remaining specimens will be destroyed. 
 
 
 
4. STATISTICAL CONSIDERATIONS/DATA ANALYSIS 
 
4.1   Statistical Calculations 
Although existing data identifies a positive correlation between ANH and a reduced need for 
allogenic transfusion, the procedures effect on markers of coagulation and platelet activation in 
patients undergoing CABG surgery remains untested. As such a reasonable calculation of a          
sample size based on the accepted error rate is currently not feasible. 
      
4.2   Statistical Analysis 
Categorical variables will be expressed as n (%). Continuous variables will be reported as 
mean±SD, or median (interquartile range), where appropriate. The paired t-test or Wilcoxon 
signed rank test or will be performed to test differences in lab values before and after treatment. 
Correlation coefficients will be performed by Pearson’s or Spearman’s Rho, where appropriate. 
Predictive cut-point values of platelet function tests to clinical endpoints will be assessed by 
ROC curve analysis with area under curve (AUC) statistics and 95% confidence intervals 
presented. Although underpowered given the pilot nature, to address the hypothesis that ANH 
will be associated with changes in hemostatic markers, reduced chest-tube drainage, reduction in 
transfusion, etc., we will use separate repeated measures generalized linear mixed model 
(GLMM) with a random intercept and the appropriate link function (e.g., binary, logit, etc.). 
Statistical calculations will be carried out using SAS (ver. 9.3, Cary, NC).  
 
4.3   Data Storage 
 
4.3.1 Data Management 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). 
Each subject screened and enrolled will be assigned a subject identification number (SID) 
and a list of subjects with their corresponding SID will be maintained separately from 
collected data. Physical CRFs will be stored at the research site in a locked office and 
electronic subject data will be locked in a password protected file on a secure internet 
server, accessed only by authorized research staff.   
 
4.3.2 Records Retention 

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The Investigator will maintain the records of final CRFs (CDROM copies), worksheets, 
source documents, and all other study-specific documentation in accordance with ICH 
Guidelines. Essential documents should be retained for at least three (3) years after the 
investigation is formally discontinued. 
 
 
5. HUMAN SUBJECTS PROTECTION (Risks, Benefits, and Alternatives) 
 
5.1 Risks 
 
5.1.1 Potential Loss of Privacy 
Protected health information (PHI) will be collected during the study. The risk for breach of 
confidentiality and privacy will be minimized by shielding the subjects unlinking his or her 
identity from his or her personal health information. 
 
5.1.2 Potential Adverse Events 
Aside from additional phlebotomies allowing blood collection as per protocol, no other 
additional treatments or procedures will be given to subjects for this trial. One research 
venipuncture maybe necessary to obtain the 16-24 hour blood draw. As such, pain, discomfort, 
or bruising on the venipuncture site may occur.  
 
5.2 Benefits and Alternatives 
There are no direct benefits to the patient. Participation in the study is entirely voluntary. The 
alternative is not to participate in the trial but to still receive the ANH procedure as part of CABG 
surgery. 
 
6. SCHEDULE OF ASSESSMENTS 
 
6.1 Laboratory Assessments 
 Light transmittance platelet aggregation stimulated by ADP, collagen, TRAP, and 
Arachidonic acid (at all study time points). 
 VerifyNow® platelet function testing, multiple cartridges (Pre CPB, Off CPB, Post-
ANH1, and 16-24hr Post Surgery) 
 Platelet Count using Plateletworks system (Pre CPB, Off CPB, Post-ANH1, and 16- 24hr 
Post Surgery) 
 Hematocrit using GEM 3500® system (Pre CPB, Off CPB, Post-ANH1, and 16-24hr 
 Post Surgery 
 Hemoglobin using HemoCue® Hb201+ system (Pre CPB, Off CPB, Post-ANH1, and 
 16- 24hr Post Surgery 
 Thrombelastography by ROTEM® (Pre CPB, Off CPB, Post-ANH1, and 16-24 hours 
 post-surgery) 
 Calibrated Automated Thrombogram (Pre CPB, just prior to and post- ANH1) 
 Real time evaluation of thrombus formation using by novel T-TAS® plus system (Pre- 
 CPB, just prior to and Post ANH1) 

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 Fibrinogen, D-dimer, vWF and CRP (Pre CPB, immediately after ANH1, 2-4 hours 
 post-surgery, and 16-24 hours post- surgery) 
 
1 Post surgical assessments will be collected from both the experimental group and the control group. Post CPB assessments will be collected from the 
control group at the same intervals (1 hour, 2-4 hours, and 16-24 hours) as the experimental group with the exception of the post ANH and post 
protamine draw. 
 
6.2 Health and Concomitant Medication Review 
Changes in participant’s medical condition (any new diagnosis, etc.) and concomitant medications 
will be continuously updated via medical records review. Serious adverse events, and clinical 
endpoints and adverse events specified in section 7.1 will also be continuously assessed during 
hospital stay.  
 
 
6.3 Physical Examination 
The physical examination is completed as standard of care for preoperative evaluation. Data 
from the physical exam will be used to evaluate the general status of the subject and to further 
elucidate patient symptoms, risk factors, or concerns that may increase the subject’s risk for 
adverse events.  
 
6.4 Safety Assessments 
Safety assessments will consist of the monitoring and recording of serious adverse events. The 
occurrence of adverse events will be ascertained via clinical assessment of bleeding. For the 
purpose of this study, thromboembolic, bleeding events and serious adverse events (SAEs) 
directly related to cardiac surgery utilizing the CPB machine, and SAEs directly related to study 
procedures (needle stick) will be collected and reported. Additional assessments required to 
ensure the safety of subjects will be administered as deemed necessary by the investigator on a 
case by case basis.  
 
6.5 Schedule of Procedures 
The study is comprised of 3 periods: Preoperative and Screening, During Surgery, and 
Postoperative. The Preoperative and Screening period is comprised of: baseline assessments and 
assessment of coagulation markers. The During Surgery period is comprised of a baseline before 
ANH removal, 30 minutes post-CBP start, off CPB, post-protamine infusion, and post-ANH 
reinfusion timepoints. The Postoperative period is comprised of: 1 hour, 2-4 hours, and 16-24 
hours post ANH reinfusion timepoints and routine standard of care evaluation for CABG surgery 
with longitudinal follow up in STS database. Medical records will be reviewed upon hospital 
discharge to assess for adverse events and documentation of discharge date. See table below for 
timing of assessments. 
 
Study Assessment\/Procedure 
Preoperative 
During Surgery 
Postoperative 
30 day 
post- 
surgery 
follow 
up5 
Screening 
Baseline 
before 
ANH 
removal 
30 
minutes 
post CBP 
start 
Immediatel
y Off CPB  
Post 
protamine 
infusion 
before ANH 
Immediately Post 
ANH Reinfusion4 
 
1 hour post 
ANH 
(+15 
minutes) 
2-4 hours 
post ANH 
16-24 
hours post 
ANH 
Assessments 
Explain Study/ Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Review of Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
 
 
 
 
 
Medical History and 
Demographics 
X 
 
 
 
 
 
 
 
 
X 
Physical Examination 
X1 
 
 
 
 
 
 
 
 
X 
Vital Signs Assessment 
X1 
 
 
 
 
 
 
 
 
X 

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Previous and Concomitant 
Medications 
X 
 
 
 
 
 
 
 
 
X 
Adverse Events Assessment 
(Bleeding and Non-Bleeding) 
 
X 
Serious Adverse Events 
Assessment (Bleeding and Non-
Bleeding) 
 
X 
Clinical Efficacy Endpoint 
Assessment (Bleeding, 
Transfusion) 
 
X 
                                            Standard of Care Local Laboratory Tests 
Hematology2 
X1 
X 
 
 
 
 
 
X 
X 
 
PT/INR, aPTT2 
X1 
X 
 
 
 
 
 
X 
X 
 
                             Biomarker Testing 
 
Fibrinogen 
X1 
X 
 
 
 
X 
 
X 
X 
 
D-Dimer 
X1 
X 
 
 
 
X 
 
X 
X 
 
Von Willebrand Factor 
X1 
X 
 
 
 
X 
 
X 
X 
 
C Reactive Protein 
X1 
X 
 
 
 
X 
 
X 
X 
 
                                       Platelet Function Testing 
 
Light transmittance aggregation 
 
X 
X3 
X 
X 
X 
X 
X 
X 
 
VerifyNow  
 
X 
 
X 
 
X 
 
 
X 
 
Thrombelastography by ROTEM 
 
X 
 
X 
 
X 
 
 
X 
 
Platelet count by Plateletworks  
 
X 
 
X 
 
X 
 
 
X 
 
Hemoglobin by Hemocue 
 
X 
 
X 
 
X 
 
 
X 
 
Hematocrit by GEM 3500 
 
X 
 
X 
 
X 
 
 
X 
 
Calibrated Automated 
Thrombogram 
 
X 
 
 
X 
X 
 
 
 
 
Real time evaluation of thrombus 
formation T-TAS 
 
X 
 
 
X 
X 
 
 
 
 
 
1 Data from the preoperative physical exam completed as per standard of care will be collected  
2 Standard of care laboratory measurements 
3 Laboratory collections only from autologous blood collected4 Post ANH assessments will be collected from the experimental group. In the control 
group the ‘immediately post ANH reinfusion  assessment will not be done. 
5 Routine post discharge standard of care office evaluation. Longitudinal evaluation follow- up in STS database 
 
 
6.5.1 Timing of Assessments  
6.5.1.1 Preoperative/Screening 
Subjects will be pre-screened to ensure that the subject is eligible for the study. All study 
inclusion criteria must be met and subject must not meet any exclusion criteria. During 
the screening visit, the following will be completed and documented in the Case Report 
Forms (CRF): 
• 
Obtain written informed consent 
• 
Complete inclusion/exclusion criteria 
• 
Obtain demographic information (i.e. date of birth, gender, and race) 
• 
Record medical history  
• 
Record concomitant medications 
• 
Obtain data from pre-operative physical exam, vital signs, height and weight 
 
completed as standard of care  
• 
Obtain pre-operative hematology and PT/INR, aPTT results (standard of care 
 
laboratory measurements) (See table) 
• 
Complete blood count, Fibrinogen, D-Dimer, von Willebrand factor, and C reactive 
 
protein may be collected at screening as long as CABG surgery is less than seven 
 
days from the collection date. These assessments will be collected on the day of 
 
surgery during the ‘Baseline’ assessment if screening exceeds the surgery by more 
 
than seven days. 
 
6.5.1.2 Perioperative Period:  
6.5.1.3 Baseline  

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Study will begin when the baseline study hematocrit assessment blood sample drawn in 
the CVOR in the experimental group and post CPB in the control group. Baseline labs 
are assed in the experimental ANH recipient group before ANH removal, 30 minutes 
post-CBP start, off CPB, post-protamine infusion, and post-ANH reinfusion timepoints. 
Baseline labs are assed in the control non- ANH group before ANH removal, 30 minutes 
post-CBP start, and off CPB timepoints.   During this period, the following procedures 
will be completed: 
•       Laboratory measurements collection of markers of coagulation and platelet    
 
function. (See Schedule of Assessments for specific laboratory assays needed for 
 
each timepoint) 
 
6.5.1.4 Postoperative Period:  
Blood drawn immediately post completion of ANH reinfusion will define the time point 
that  all post-operative blood draws in the experimental group will be based on, while off 
CPB will define the time point that all post-operative blood draws in the control group 
will be based on. Time points for the postoperative period include 1 hour (+ 15 minutes), 
2-4 hours, and 16-24 hours post-surgery. The following study procedures will be 
completed: 
•         Laboratory measurements collection (see table of assessments for specific    
 
   laboratory assays needed for each timepoint) 
• 
   Record total CT drainage amount in 24 hours post placement 
• 
   Record total number of whole blood or packed red cells transfused, if applicable.  
 
6.5.1.5 Hospital discharge and Follow up:  
Patient will undergo routine post-operative discharge evaluation that is standard of care 
for all CABG surgeries, and longitudinal follow up in STS database. Patient post- 
operative evaluation will include all standard of care assessments as well as occurrence 
of adverse events, serious adverse events and clinical efficacy assessments related to 
bleeding and total number of blood products infused. 
 
7. ADVERSE EVENTS   
 
7.1 AE Recording 
Adverse events will be collected postoperatively. For the purpose of this study, only the 
following adverse events will be collected and reported: 
     SAE’s related to study procedures (i.e. SAE’s related to phlebotomy procedure) 
7.2 Adverse Events Treatment 
All AEs should be treated appropriately and managed according to standard of care. The        
action taken to treat the AE should be recorded on the AE CRF. Assessment should be           
made at each protocol time point of any changes in severity, relationship to the study                 
procedures (if applicable), and the interventions required to treat it, and the outcomes of those 
interventions. 
 
7.3 Adverse Event Documentation   

Page 14 of 18 
 The principal investigator has the primary responsibility for SAE identification,  
documentation, grading, and assignment of attribution to the study intervention. SAEs must be 
recorded in the AE CRF with the following information:  
      AE term 
      The intensity grade (CTCAE v 4.03 grading) 
      The relationship to study procedure 
      Attribution 
      Duration 
      Occurrence (known risks for study procedure-expected, unexpected) 
      Other contributing causes 
      Actions in response to event 
      Outcome 
      Criteria for SAE  
 
7.4 
AE Grading Scale 
The descriptions and grading scales found in NCI Common Terminology Criteria for Adverse  
Events (CTCAE) version 4.03 will be used for AE reporting. Each AE term is associated with a 
5-point severity scale.  
 
7.5 Protocol Deviations and Violations 
The Principal Investigator will not deviate from the protocol without obtaining approval            
from the IRB or Ethics Committee and the sponsor. Protocol deviations that occur will be          
reported to the Inova IRB at the time of continuing review. Protocol violations that affect          
the subject’s rights and safety, and/or affects study integrity will be reported to the Inova           
IRB within 10 working days of event knowledge.  
 
8. STUDY COST 
The subject or their insurance company will not be billed for this study. All study related tests 
that is not considered as standard of care will be paid for by the research site. 
9. FUNDING 
This is an investigator-initiated study sponsored by Inova Heart and Vascular Institute and 
Instrumentation Laboratories, Inc. There is no investigational product involved. Treatment 
received by the subject will be as according to main provider’s discretion as according to 
standard of care. 
 
10. CONFLICTS OF INTEREST   
The investigators declare no conflicts of interest linked to this study.  
 
11. FACILITIES AND EQUIPMENT  
The research site is equipped with its own laboratory equipment, which includes state of               
the art technologies for platelet assays, centrifuges, refrigerators, and freezers for                          
study specimen processing and storage.  
 

Page 15 of 18 
 
12. OUTSIDE CONSULTANTS/COLLABORATORS  
There are no outside consultants/collaborators participating. 
 
13. CONTRACTURAL AGREEMENTS     
There are no outside consultants/collaborators participating. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Page 16 of 18 
 
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