Nattokinase Atherothrombotic Prevention Study
Phase: NA
Conditions: Prevention of Subclinical Atherosclerosis Progression, Prevention of Cognitive Decline
Sponsor: University of Southern California

 
 
 
 
 
 
 
NATTOKINASE ATHEROTHROMBOTIC PREVENTION STUDY (NAPS) 
 
NCT 02080520 
 
Manual of Operations 
 
March 10, 2014 
 
 
 
 

 
Version: 03-10-14 
 
1 
 
 
 
Table of Contents 
 
 
 
NATTOKINASE ATHEROTHROMBOTIC PREVENTION STUDY (NAPS) 
 
 
 
 
Manual of Operations 
 
 
 
 
 
 
1.0 
Overview/Eligibility..................................................................................................... 2 
 
2.0 
Organizational Structure.............................................................................................. 
9 
 
3.0 
Recruitment/Screening................................................................................................ 
11 
 
4.0 
Randomization............................................................................................................ 
14 
 
5.0 
Compliance and Dropout............................................................................................. 
17 
 
6.0 
Clinical Events and Definitions.................................................................................... 18 
 
7.0 
Laboratory Protocol.................................................................................................... 
24 
 
8.0 
Carotid Ultrasonographic Protocol.............................................................................. 
46 
 
9.0 
Data Collection and Management................................................................................ 64 

Manual of Operations 
 
 
Version: 03-10-14 
 
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1.0 
 
OVERVIEW/ELIGIBILITY 
 
1.1  Executive Summary 
 
The potential for nattokinase to “thin” blood and to reduce blood clotting by positive antithrombotic and 
fibrinolytic effects presents a unique opportunity to safely study such effects on cardiovascular disease 
(CVD) and cognition. Unfortunately, such studies of antithrombotic and fibrinolytic pathways of 
prevention have been limited due to lack of safe compounds and the adverse reactions associated with 
current agents such as Coumadin. Nattokinase, an over-the-counter supplement used for cardiovascular 
health, is the most active functional constituent of natto, a fermented soy product. Natto has been 
consumed primarily by the Japanese for over 1000 years, a population with one of the lowest risks for 
CVD and dementia. CVD and dementia remain the most challenging age-related health risks of the 21st 
century for Americans necessitating development of further effective preemptive strategies. Whether 
reducing the propensity for thrombus formation and/or increasing fibrinolytic activity can prevent the 
progression of atherosclerosis and cognitive decline has not yet been determined. 
 
Using nattokinase under primary prevention conditions, we propose to conduct a randomized, double-
blinded, placebo-controlled trial to determine whether decreasing atherothrombotic risk can reduce the 
progression of atherosclerosis and cognitive decline. We propose to randomize 240 healthy non-
demented women and men to nattokinase supplementation or to placebo for three years. The primary 
trial end points will be measurement of carotid arterial wall thickness and arterial stiffness, early 
changes of atherosclerosis that can be measured safely by non-invasive imaging techniques. The 
secondary trial end point will be ascertained through change in cognition measured by a 
neuropsychological battery. In addition, biochemical blood measurements and in vitro studies will be 
conducted to compare the effects of nattokinase relative to placebo on blood coagulation and thrombus 
break-down capabilities, blood flow properties, inflammation and inflammatory activation of endothelial 
cells that line blood vessels. 
 
At the conclusion of this trial, we expect to have sufficient evidence as to whether reducing propensity 
for thrombus formation and/or increasing fibrinolytic activity can prevent atherosclerosis progression 
and cognitive decline. Results will provide novel and important data that will be informative concerning 
primary prevention through the atherothrombotic pathway. Providing evidence for a reduction in 
atherosclerosis progression and cognitive decline with nattokinase is likely to shift the current clinical 
paradigm for the prevention of these chronic age-related processes. Additionally, such evidence will 
serve to create a new field of discovery and opportunity for prevention of CVD and dementia. 
 
1.2  Objectives and Hypotheses 
 
The goal of the proposed study is to determine under randomized controlled trial conditions whether 
nattokinase reduces subclinical atherosclerosis and cognitive decline in healthy women and men. Our 
hypotheses are: 1) Compared to placebo, nattokinase will show less subclinical atherosclerosis 
progression and cognitive decline in healthy women and men; 2) The reduction in subclinical 
atherosclerosis progression and cognitive decline with nattokinase will be correlated; and, 3) The 
reduction in progression of subclinical atherosclerosis and cognitive decline with nattokinase will be 
mediated through hemostatic, fibrinolytic and hemorheological factors as well as attenuation of 
inflammation, monocyte activation, vascular endothelium injury and activation of vascular endothelium 
by circulating monocytes. 
 

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1.3  Study Overview 
 
The Nattokinase Atherothrombotic Prevention Study (NAPS) is a randomized, double-blinded, 
placebo-controlled, non-invasive vascular imaging trial. A total of 240 healthy non-demented women 
and men without clinical evidence of CVD or diabetes mellitus will be randomized to either placebo or 
to active oral nattokinase 2,000 fibrinolytic units daily. Recruitment will occur over 2 years and the 
treatment period will be for 3 years. Candidates will be prescreened, seen for 1 screening visit to 
determine eligibility and then randomized at a baseline visit. Participants will then return for a follow-up 
visit 1 week after randomization and then will be followed monthly for the first 6 months and then every 
3 months for the remainder of the trial. Rate of change in distal common carotid artery (CCA) far wall 
intima-media thickness (CIMT) and arterial stiffness in computer image processed B-mode 
ultrasonograms will be the co-primary trial end points. Cognitive decline determined with a 
neuropsychological battery designed to evaluate 7 cognitive domains including: attention, concentration, 
working memory, executive function; visuospatial/visuoconstructive skills; naming/semantic memory; 
and verbal and nonverbal episodic memory will be the secondary trial endpoint. Other endpoints will 
include blood pressure, hemostatic, fibrinolytic and hemorheological factors as well as platelet activity 
and biomarkers for inflammation, monocyte activation, vascular endothelium injury and activation of 
vascular endothelium by circulating monocytes. 
 
1.4  Inclusion Criteria 
 
(1) 
Age >55 years 
(2) 
Male or postmenopausal female (no uterine bleeding for >6 months) 
 
1.5  Exclusion Criteria 
 
(1) 
Clinical signs, symptoms, or personal history of CVD 
(2) 
Diabetes mellitus or fasting serum glucose >140 mg/dL 
(3) 
Plasma triglyceride levels >500 mg/dL 
(4) 
Uncontrolled hypertension (systolic blood pressure >160 mmHg or diastolic blood pressure >110 
mmHg)* 
(5) 
Uncontrolled tachycardia or irregular heart rates (i.e., atrial fibrillation) 
(6) 
Thyroid disease (untreated) 
(7) 
Renal insufficiency (defined as serum creatinine >2.0 mg/dL) 
(8) 
Life threatening illness with prognosis <5 years 
(9) 
Current use of lipid-lowering medication 
(10) 
Current use of food supplements containing soy, soy protein, isoflavones or other phytoestrogens 
(11) 
Known sensitivity or allergy to soy or nuts 
(12) 
Regular aspirin or other antiplatelet medication use 
(13) 
Use of anticoagulants 
(14) 
Bleeding diatheses or tendencies 
 
*Participants who enter screening with a systolic blood pressure >160 mmHg or diastolic blood pressure 
>110 mmHg will be immediately referred to their physician or to an emergency room. Once the blood 
pressure is controlled, the participant will become eligible for the trial. Participants who enter screening 
with mild to moderate hypertension (systolic blood pressure <160 mmHg or diastolic blood pressure 
<110 mmHg) will be notified immediately and a letter issued to the participant and to the participant's 
physician. Control of hypertension is a requisite to continue into the trial. 

Manual of Operations 
 
 
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1.6 
Trial Clinic Flow 
 
Recruitment 
Sources 
⇓ 
Prescreening 
- Telephone Questionnaire [Form 1] 
 
 
 
 
 
 
 
⇓ 
- Age >55 years 
 
 
 
 
 
 
 
⇓ 
- Male or postmenopausal female 
 
 
 
 
  
   
 
 
⇓ 
- No cardiovascular disease  
 
 
 
 
 
 
  
   
 
 
⇓ 
- No diabetes mellitus 
 
 
 
 
  
   
 
 
⇓ 
- No life threatening disease  
 
 
 
 
 
 
  
   
 
 
⇓ 
- No current use of lipid-lowering medication 
 
 
 
  
   
 
 
⇓ 
- No current use of food supplements containing soy, 
 
 
 
  
   
 
 
 
 soy protein, isoflavones or other phytoestrogens 
⇓ 
- No known sensitivity or allergy to soy or nuts 
⇓ 
- No regular use of aspirin or other antiplatelet 
 
 
 
 
  
   
 
 
 
 medication 
⇓ 
- No use of anticoagulants 
⇓ 
- No bleeding diatheses or tendencies 
 
  
   
 
 
⇓ 
If taking, instruct individual to stop soy, soy protein and isoflavone food supplements 
for 1 month prior to screening visit 
 
 
 
 
  
   
 
 
⇓ 
 
 
 
 
  
   
 
 
⇓ 
Schedule screening visit appointment 
8 hour fast 
 
 
 
 
  
   
 
⇓ 
 
 
 
 
  
   
 
 
⇓ 
Screening Visit 
- Assign study ID 
- Informed consent and HIPAA [Form 2] 
- Participant Information Form [Form 3] 
- Personal Information Form [Form 4] 
- Cardiovascular Disease History Questionnaire [Form 5] 
- Bleeding Assessment Questionnaire [Form 6a] 
- Smoking/ETOH Questionnaire [Form 7a] 
- Reproductive History Questionnaire [Form 8] 
- Medical History Questionnaire [Form 9] 
- Rose Questionnaire [Form 10] 
- Claudication Questionnaire [Form 11] 
- Cardiovascular Disease Risk Factor Questionnaire [Form 12] 
- Nonstudy Medications Record [Forms 13,14] 
- History of Vitamin and Nutritional Supplement Use Questionnaire [Form 15] 
- Vital signs (systolic blood pressure <160 mmHg and diastolic blood pressure 
 <110 mmHg) [Form 16] 
- Pulse rate [Form 16] 
- Weight/height [Form 16] 
- CIMT-1/arterial stiffness 
 
 

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- Fasting blood samples drawn for: 
  
  
- Chemistry (FSG <140 mg/dL; serum creatinine <2.0 mg/dL) 
- CBC 
- Platelet count 
- Prothrombin time (PT) 
- activated partial thromboplastin time (aPTT) 
- Lipids (triglycerides <500 mg/dL) 
- C-reactive protein (CRP) 
- Hemoglobin A1c 
 
 
 
 
Draw and store at -80oC (analyze only when eligible): 
- Insulin 
- Inflammatory markers 
   
- MCP-1 
   
- IL-8 
   
- TNFα 
   
- IL-1β 
   
- IL-10 
   
- E-Selectin 
   
- P-Selectin 
 
⇓ 
 
⇓ 
Schedule randomization visit appointment 
8 hour fast 
 
⇓ 
 
⇓ 
Final Eligibility 
- Final Eligibility Check List [Form 17] 
 
⇓ 
 
⇓ 
Randomization Visit (v00) 
- Nonstudy Medications Record [Forms 13,14] 
- History of Vitamin and Nutritional Supplement Use Questionnaire [Form 15] 
- Dietary Food Frequency [Form 18] 
- Soy Food Questionnaire [Form 19] 
- 7-Day Physical Activity Questionnaire [Form 20] 
- Center for Epidemiologic Studies Depression (CES-D) Scale [Form 21] 
- Cognitive assessment [Form 22] 
- Vitals signs (systolic blood pressure <160 mmHg and diastolic blood pressure 
 <110 mmHg) [Form 16] 
- Pulse rate [Form 16] 
- Weight [Form 16] 
- Waist:hip measurement [Form 23]  
- 12-lead ECG [Form 24] 
- CIMT-2/arterial stiffness 
- Fasting blood samples drawn for: 
- Fibrinogen 
- vWF antigen 
- Tissue plasminogen activator (tPA) 
- Plasminogen activator inhibitor-1 (PAI-1) 
- D-dimers 
- Platelet aggregation (ADP, collagen, epinephrine) 
- Ristocetin cofactor 
- Factor VIII 

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- Nattokinase 
- Anti-nattokinase antibodies 
- Erythrocyte sedimentation rate (ESR) 
- Red blood cell aggregation (plasma) 
- Red blood cell aggregability (polymer) 
- Plasma viscosity 
- Whole blood viscosity 
- Endothelial injury in-vitro assay: VCAM-1, ICAM-1 
- Monocyte activation (FACS): CD11b, CD11c, VLA-4 
- Storage 
   
Plasma (EDTA) generic storage 
Plasma (citrate) generic storage 
Serum generic storage 
Buffy Coat 
 
 
 
 
  
- Perform urine dipstick 
- Randomization based on CIMT-1 <>0.75 mm 
- Active nattokinase/placebo nattokinase dispensed [Form 25] 
    
  
   
 
⇓ 
 
⇓ 
Schedule week 1 appointment 
8 hour fast 
 
  
   
 
⇓ 
 
⇓ 
Week 1 
- Bleeding Assessment Questionnaire [Form 6b] 
- Fasting blood samples drawn for: 
- Fibrinogen 
- vWF antigen 
- Tissue plasminogen activator (tPA) 
- Plasminogen activator inhibitor-1 (PAI-1) 
- D-dimers 
- Ristocetin cofactor 
- Factor VIII 
- Nattokinase 
- Erythrocyte sedimentation rate (ESR) 
- Red blood cell aggregation (plasma) 
- Red blood cell aggregability (polymer) 
- Plasma viscosity 
- Whole blood viscosity 
- Storage 
Plasma (EDTA) generic storage 
Plasma (citrate) generic storage 
Serum generic storage 
 
 

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1.7  Schedule of Visits and Laboratory Studies (See Section 7.6 (Table) for specific details) 
 
 
 
 
 
MONTH 
 
 
RESEARCH ACTIVITY 
SCREEN 
BASELINE 
WEEK 1 
 
 
 
01 
 
 
 
02 
 
 
 
03 
 
 
 
04 
 
 
 
05 
 
 
 
06 
 
 
 
09 
 
 
 
12 
 
 
 
15 
 
 
 
18 
 
 
 
21 
 
 
 
24 
 
 
 
27 
 
 
 
30 
 
 
 
33 
 
 
 
36 
Chemistry Panel 
x 
 
 
x 
x 
x 
x 
x 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Insulin 
x 
 
 
 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Complete Blood Count 
x 
 
 
x 
x 
x 
x 
x 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Platelet Count 
x 
 
 
x 
x 
x 
x 
x 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Prothrombin time (PT) 
x 
 
 
x 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Activated Partial Thromboplastin Time (aPTT) 
x 
 
 
x 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Fibrinogen 
 
x 
x 
x 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
vWF Antigen 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Tissue Plasminogen Activator (tPA) 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Plasminogen Activator Inhitor-1 (PAI-1) 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
D-Dimers 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Platelet Aggregation (ADP, Collagen, Epinephrine) 
 
x 
 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
Ristocetin Cofactor 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Factor VIII 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Nattokinase 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Anti-Nattokinase Antibodies 
 
x 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Erythrocyte Sedimentation Rate (ESR) 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Red Blood Cell Aggregation (Plasma) 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Red Blood Cell Aggregability (Polymer) 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Plasma Viscosity 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Whole Blood Viscosity 
 
x 
x 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
MCP-1 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
IL-8 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
TNFα 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
IL-1β   
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
IL-10 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
E-Selectin 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
P-Selectin 
x 
 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
Endothelial Injury In-Vitro Assay: VCAM-1, ICAM-1 
 
x 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
Monocyte Activation (FACS): CD11b, CD11c, VLA-4 
 
x 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
Lipids/C-Reactive Protein (CRP) 
x 
 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Hemoglobin A1c 
x 
 
 
 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Storage 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Buffy Coat 
 
x 
 
 
x 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
ApoE Genotype 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Dipstick 
 
x 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Manual of Operations 
 
 
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8 
 
1.8  Schedule of Visits, Questionnaires and Procedures 
 
 
 
 
 
 
 
MONTH 
 
 
 
RESEARCH ACTIVITY 
FORM 
NUMBER 
PRESCREEN 
SCREEN 
BASELINE 
WEEK 1 
 
 
 
01 
 
 
 
02 
 
 
 
03 
 
 
 
04 
 
 
 
05 
 
 
 
06 
 
 
 
09 
 
 
 
12 
 
 
 
15 
 
 
 
18 
 
 
 
21 
 
 
 
24 
 
 
 
27 
 
 
 
30 
 
 
 
33 
 
 
 
36 
Telephone Questionnaire 
1 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assign Study ID 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
2 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participant Information 
3 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Personal Information 
4 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cardiovascular Disease History 
5 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bleeding Assessment Questionnaire 
6a, 6b 
 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Active/Passive Smoking Questionnaire 
7a, 7b 
 
x 
 
 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
Reproductive History (Female) 
8 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History Baseline 
9 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Rose Questionnaire 
10 
 
x 
 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Claudication Questionnaire 
11 
 
x 
 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Cardiovascular Risk Factor Questionnaire 
12 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Non-study Medications 
13,14 
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Nutraceuticals and Supplements 
15 
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Blood Pressure 
16 
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Pulse Rate 
16 
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Weight 
16 
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Final Eligibility Checklist 
17 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dietary Food Frequency 
18 
 
 
x 
 
 
 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
Soy Food Questionnaire 
19 
 
 
x 
 
 
 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
7-Day Physical Activity Recall 
20 
 
 
x 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
CES-D* 
21 
 
 
x 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Cognitive Assessment 
22 
 
 
x 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
x 
Waist:Hip Circumference 
23 
 
 
x 
 
 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
12-Lead Electrocardiogram 
24 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
Dispense Study Products 
25 
 
 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Randomize Participant 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Compliance 
26 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Medication Termination/Dropout Form 
27** 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse Events 
28-38 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Medical History On-Trial 
39 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
Carotid Artery Intima-Media Thickness 
 
 
x 
x 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Arterial Stiffness 
 
 
x 
x 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
Closeout Form 
40 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
*CES-D = Center for Epidemiologic Studies Depression Scale 
**Use as needed 
 

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2.0 
 
ORGANIZATIONAL STRUCTURE 
 
The organizational structure is described in terms of five units according to their function in the overall 
study. In addition, there is a Steering Committee and an External Data and Safety Monitoring Board 
(EDSMB). 
 
1)  Administration 
 
2)  Clinic 
a) Physician 
b) Study coordinator 
c) Research nurses 
d) Phlebotomists 
e) Participant coordinators 
f) Recruiters 
g) Cognitive tester 
 
3)  Laboratory 
a) Core lipid laboratory – USC  
b) Hematology laboratory – USC  
c) Rheology laboratory – USC  
d) Biochemistry laboratory – USC 
e) Quest (Los Angeles) 
 
4)  Core ultrasound imaging facility 
 
5)  Data Coordinating Center (DCC) 
a) Biostatisticians 
b) Programmers 
c) Data clerks 
 
These units interact directly under the coordination of the principal investigator. In addition, there are 
two oversight bodies: 
 
1)  Institutional Review Board (IRB) 
2)  External Data and Safety Monitoring Board (EDSMB) 
 
The Steering Committee is comprised of the following individuals: 
 
Howard N. Hodis, M.D., Chairman 
Wendy J. Mack, Ph.D., Co-Chair 
Herbert J. Meiselman, Sc.D. 
Howard A. Liebman, M.D. 
Vijay Kalra, Ph.D. 
 
 
 
 

Manual of Operations 
 
 
Version: 03-10-14 
 
10 
The External Data and Safety Monitoring Board is comprised of the following individuals: 
 
Chairman 
 
Christopher J. Gallagher, M.D. 
Professor of Medicine 
Creighton University School of Medicine 
 
Dennis M. Black, Ph.D. 
Professor in Residence 
Division of Clinical Trials and Multicenter Studies 
University of California, San Francisco 
 
Kenneth A. Bauer, M.D. 
Professor of Medicine 
Hematology-Oncology 
Harvard Medical School 
 
 
 
 
 
 

Manual of Operations 
 
 
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11 
 
3.0 
 
RECRUITMENT/SCREENING 
 
3.1  Recruitment 
 
Potential participants will be recruited from multiple sources, including the University of Southern 
California. 
 
We plan to target recruitment of 50% women and 30% minorities for this trial (see Table below). 
 
 
 
American 
Indian or 
Alaskan Native 
 
 
Asian or Pacific 
Islander 
 
Black, not 
of Hispanic 
Origin 
 
 
 
Hispanic 
 
White, not 
of Hispanic 
Origin 
 
 
 
Total 
 
Female 
 
1 
 
6 
 
11 
 
18 
 
84 
 
120 
 
Male 
 
1 
 
6 
 
11 
 
18 
 
84 
 
120 
 
Total 
 
2 
 
12 
 
 
22 
 
36 
 
 
168 
 
240 
 
3.2  Screening 
 
Potential participants will be pre-screened with a Telephone Questionnaire [FORM 1] that will cover 
major inclusion and exclusion criteria. The study will be explained to potential participants at this time. 
 
Individuals fulfilling the Telephone Questionnaire eligibility criteria and interested in the study will be 
scheduled for the screening visit (SV) and instructed to stop soy, soy protein and isoflavone food 
supplements one month prior to SV if currently taking any of these products. 
 
Participants will be instructed to fast for 8 hours prior to SV. 
 
Final study eligibility will be determined over SV and the baseline visit (v00). 
 
      1 week* 
  2 weeks 
Telephone pre-screening             SV           v00 (randomization) 
 *1 month if potential participant needs to stop soy, soy protein and isoflavone food supplement  
 
 
        
intake prior to SV.
 
3.3  Screening Visit (SV) 
 
A SV appointment is contingent upon fulfilling all pre-screening eligibility criteria [FORM 1]. At SV, 
the following will be performed: 
 
 1. 
Assign study identification (ID) number (see Section 3.4 for procedure) 
 2. 
Explanation of study aims, protocol, benefits, risks 
 3. 
Participants sign Informed Consent and HIPPA [FORM 2] 
 4. 
Participants complete forms covering contact and demographic information: 
Participant Information Form [FORM 3] 
Personal Information Form [FORM 4] 
 

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12 
 5. 
Pre-screening Telephone Questionnaire [FORM 1] eligibility criteria reviewed with participant 
 6. 
Ensure that soy, soy protein and isoflavone food supplements have been stopped at least one 
month prior to SV (see Section 3.2) 
 7. 
Administer the Cardiovascular Disease History Questionnaire [FORM 5] and confirm that 
participants have no clinical manifestations of cardiovascular disease. 
 
 8. 
Administer the baseline Bleeding Assessment Questionnaire [FORM 6a] and confirm that 
participants have no bleeding diatheses or bleeding tendencies 
 9. 
Participants administered baseline Smoking, Passive Smoking, Alcohol, Other Substance Use 
 
Questionnaire [FORM 7a] 
10. Administer the Reproductive History Questionnaire [FORM 8] and confirm that female 
participants have not menstruated for at least 6 months 
11. Participants administered questionnaires covering health information: 
Medical History Questionnaire [FORM 9] 
Modified Rose Questionnaire [FORM 10] 
Claudication Questionnaire [FORM 11] 
 
 
Cardiovascular Disease Risk Factor Assessment Form [FORM 12] 
12. Administer the Nonstudy Medications Record [FORMS 13,14] and confirm that participants are 
not taking lipid-lowering medication 
13. Administer History of Vitamin and Nutritional Supplement Use Questionnaire [FORM 15] and 
confirm that participants are not taking soy, soy protein and isoflavone food supplements 
14. Obtain participant’s blood pressure and pulse rate [FORM 16] and confirm that systolic blood 
pressure is <160 mmHg and diastolic blood pressure is <110 mmHg 
15. Obtain and record participant’s weight and height [FORM 16] 
16. Carotid artery ultrasound examination performed (first baseline - see Section 4.2 for randomization 
procedure) 
17. Fasting blood samples drawn and analyzed for: 
 
 
 
 
  
- Chemistry 
 
 
 
 
  
- CBC 
- Platelet count 
- Prothrombin (PT)/activated partial thromboplastin time (aPTT) 
- Lipids 
- C-reactive protein 
- Hemoglobin A1c 
18. Fasting blood samples drawn and not analyzed until found eligible: 
- Insulin 
- Inflammatory markers 
  - MCP-1 
  - IL-8 
  - TNFα 
  - IL-1β 
  - IL-10 
  - E-Selectin 
- P-Selectin 
 
Baseline (randomization) visit (v00) is scheduled two weeks after SV to allow adequate sampling of 
baseline chemistry, CBC, platelet count, PT, aPTT, lipids/CRP and hemoglobin A1c. Participants will 
be instructed to fast for 8 hours prior to v00. 
 
 
 

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3.4  Assigning Study Identification (ID) Numbers 
 
When a participant begins the screening process, a study ID will be assigned. This study ID will remain 
with the participant throughout the study, whether or not they continue into the randomization phase of 
the trial. All study data forms and communications with the DCC will use this study ID. 
 
The study ID will be assigned by the ARU clinic personnel who make the SV clinic appointments by the 
following procedure: 
 
When the participant appears for SV, obtain the list entitled "Nattokinase Atherothrombotic Prevention 
Study (NAPS) IDs." 
 
Using the next available blank line, write in a study ID in sequence from the study ID above and the first 
three letters of the participant's last name and the first letter of the participant's first name and write the 
participant's name in the line next to the study ID. 
 
For example, Jenny Jones is found to be potentially eligible and appears for SV at the ARU research 
clinic. On the " Nattokinase Atherothrombotic Prevention Study (NAPS) IDs" sheet, it is found that the 
last assigned study ID was 8105, assigned to Mary Smith. The blank line below would be completed as 
8106JONJ and Jenny Jones written next to it. Jenny's study ID is now 8106JONJ. 
 
Write the assigned study ID in item 23 of the Telephone Questionnaire [FORM 1] and send the form to 
the DCC for data entry. In the example above, item 23 of Jenny Jones' Telephone Questionnaire would 
be completed as 8106JONJ. 
 

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4.0 
 
RANDOMIZATION 
 
4.1  Visit 00 (v00) 
 
 1. 
Complete and review Final Eligibility Check List [FORM 17] and ensure that all eligibility 
criteria have been fulfilled before proceeding with randomization 
 2. 
Administer the Nonstudy Medications Record [FORMS 13,14] and confirm that participants are 
not taking lipid-lowering medication 
 3. 
Administer History of Vitamin and Nutritional Supplement Use Questionnaire [FORM 15] and 
confirm that participants are not taking soy, soy protein and isoflavone food supplements 
 4. 
Administer Dietary Food Frequency [FORM 18] 
 5. 
Administer Soy Food Questionnaire [FORM 19] 
 6. 
Administer 7-Day Physical Activity Recall Questionnaire [FORM 20] 
 7. 
Administer Center for Epidemiologic Studies Depression (CES-D) Scale [FORM 21] 
 8. 
Perform cognitive function assessment [FORM 22] 
 9. 
Obtain participants’ blood pressure and pulse rate [FORM 16] and confirm that systolic blood 
pressure is <160 mmHg and diastolic blood pressure is <110 mmHg 
10. Obtain and record participant’s weight [FORM 16] 
11. Measure and record waist:hip circumferences [FORM 23] 
12. Obtain 12-lead ECG [FORM 24] 
13. Carotid artery ultrasound examination performed (second baseline - see Section 4.2 for 
randomization procedure) 
14. Perform urine dipstick 
15. Fasting blood samples drawn and analyzed for: 
  
- Fibrinogen 
- vWF antigen 
- Tissue plasminogen activator (tPA) 
- Plasminogen activator inhibitor-1 (PAI-1) 
- D-dimers 
- Platelet aggregation (ADP, collagen, epinephrine) 
- Ristocetin cofactor 
- Factor VIII 
- Nattokinase 
- Anti-nattokinase antibodies 
- Erythrocyte sedimentation rate (ESR) 
- Red blood cell aggregation (plasma) 
- Red blood cell aggregability (polymer) 
- Plasma viscosity 
- Whole blood viscosity 
- Endothelial injury in-vitro assay: VCAM-1, ICAM-1 
- Monocyte activation (FACS): CD11b, CD11c, VLA-4 
- Storage 
   
Plasma (EDTA) generic storage 
Plasma (citrate) generic storage 
Serum generic storage 
Buffy coat 
 
16. Active nattokinase/placebo nattokinase dispensed [FORMS 25] 

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17. Fill in the study product ID number item of the Eligibility Summary section on the Final   
  
Eligibility Check List [FORM 17] according to the randomization procedures in Section 4.2. 
 
A follow-up clinic visit will be scheduled one week following v00. Subsequent clinic visits will be 
scheduled monthly (from date of randomization) for the first six months and then every three months for 
the remainder of the trial. Participants will be instructed to fast for 8 hours prior to each clinic visit. 
 
4.2  Randomization Procedure 
 
Active nattokinase and matching nattokinase placebo will be shipped directly to the DCC by Jarrow 
Formulas. Active nattokinase and matching nattokinase placebo will be shipped on different days 
separated in time by at least one week. Jarrow Formulas will clearly label the containers as active 
nattokinase and nattokinase placebo. Active nattokinase and matching nattokinase placebo capsules will 
be sealed in white plastic bottles with a quantity of 110 capsules per bottle. Each participant will be 
given adequate numbers of bottles of capsules for the period of time between clinic visits. The bottles of 
capsules will be prepared by the DCC for each randomized participant and will be dispensed at each 
clinic visit to each participant. The bottles of capsules will contain an attached label prepared with the 
following identifiers: Stratum (1,2) and a unique identification number for each bottle of capsules. The 
label will also contain space for the participant's name, visit number and date of dispensation to be filled 
in by clinic personnel when the bottle of capsules is assigned to a randomized participant. 
 
The DCC will generate randomization lists for 2 strata with a blocking factor not identified to other 
study personnel: 
 
1)  Right distal common carotid arterial far wall IMT >0.75 mm 
2)  Right distal common carotid arterial far wall IMT <0.75 mm 
 
As the Final Eligibility Check List [Form 17] is completed and a participant is found to be eligible for 
randomization, the clinic coordinator will do the following: 
 
1) 
Using the appropriate stratum (identified in the Eligibility Summary section of the Final Eligibility 
  
 Check List [Form 17], refer to the study randomization log for that stratum. 
 
2) 
The study randomization log contains a separate list of sequential study product ID numbers for 
each stratum. Note that each stratum list has a column of study product ID numbers as well as a 
blank column to list the participant study ID. On the study randomization log for the appropriate 
stratum, write the participant study ID on the next available line. Note the study product ID 
number. 
 
3) 
Write the study product ID number in the product ID number item of the Eligibility Summary  
  
section on the Final Eligibility Check List [Form 17]. 
 
4) 
From the study product storage area containing study products that have not yet been assigned to 
randomized participants, obtain the bottle of capsules with the same study product ID number 
indicated on the randomization log and written on the Final Eligibility Check List [Form 17]. 
 
5) 
For initial randomization, the bottle of capsules will contain enough capsules to last the participant 
for three study months (one bottle of capsules). Write the participant's name, visit number and date 
of dispensation on the label of the packet to be dispensed. 

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6) 
Duplicates of the study labels will be placed on the Nattokinase/Placebo Compliance Form     
[Form 25]. 
 
7) 
Send the Final Eligibility Check List [Form 17] to the DCC. 
 
Upon receipt of the Final Eligibility Check List [FORM 17], the DCC personnel will do the following: 
 
1) 
Review form for completeness and accuracy and enter the data. 
 
2) 
Run error check routine to: 
 
a)  Confirm study eligibility. 
 
b) Assure that the participant has been randomized within the appropriate stratum. 
 
c) Inform the principal investigator and study coordinator if any of the above two protocol  
 
 
 
violations has occurred. 
 
d) Generate an ideal visit schedule for the participant and send it to the ARU clinic. This ideal 
visit schedule will include ideal clinic visits every month for the first six months and then 
every three months for the remainder of the trial. 
 
e) Generate labels for laboratory samples and distribute to clinic phlebotomist and core lipid  
 
 
 
laboratory personnel. 
 
Only the right distal CCA far wall IMT at baseline will be used as a stratifying factor. Within each 
stratum, the distribution of other risk factors (specifically, LDL-C, smoking status, age and blood 
pressure) will be monitored across the two treatment groups. If imbalance is found across the two 
treatment groups, the randomization plan will be adapted to correct for this imbalance, i.e., adaptive 
randomization. These corrections will occur after the first 100 then 200 participants have been 
randomized. 
 
4.3  Dispensing Study Products During the Trial (after Randomization) 
 
1)  At each clinic visit, record the number of capsules consumed since the last study visit on the 
Compliance Calculation Form [Form 26]. The DCC will use these data to replace the appropriate 
number of study products in the participant's study product storage container. 
 
2)  From the study product storage area containing study products for participants that have already 
been randomized, which are sorted by the participant study ID, locate the participant's stored 
products. 
 
3)  From the participant's storage containers, obtain the same number of bottles of capsules that were 
consumed since the last visit (recorded on the Compliance Calculation Form [Form 26]).

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5.0 
 
COMPLIANCE AND DROPOUT 
 
5.1  Clinic Appointments 
 
Overall compliance will be determined by the number of missed regularly scheduled clinic visits. The 
visit limit window will be +14 days of the actual clinic visit date. Out of window visits will also be 
calculated. 
 
5.2  Medication 
 
Compliance with active nattokinase and nattokinase placebo [FORM 26] will be evaluated by capsule 
count. Compliance will be defined as >80% of capsules consumed. 
 
Percentage of active nattokinase and nattokinase placebo consumed will be determined by the formula: 
 
                                   
TCD - TCR X 100, 
                                     
     D x A 
 
where TCD=total number of capsules dispensed; TCR=total number of capsules returned; D=number of 
capsules to be taken each day; and, A=actual number of days since last visit. 
 
5.3  Dropouts 
 
Participants who do not want to continue to participate in the study or to take the study product will be 
urged to complete all clinic visits and to be followed for cardiovascular disease events as well as 
undergo the remainder of carotid artery ultrasound examinations and other studies including those for 
the secondary end points. A participant who stops taking the study product but continues to attend clinic 
visits and to complete ultrasound examinations and other secondary end points will be termed a 
“medication termination.” A participant who ceases participation and does not attend clinic visits will be 
termed a “dropout.” 
 
All dropouts and reasons for terminating the study or terminating use of study product will be recorded 
on the Medication Termination/Dropout Form [FORM 27].

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6.0 
 
CLINICAL EVENTS AND DEFINITIONS 
 
6.1  Overview 
 
Participants on trial will continue with their usual source of medical care. Laboratory abnormalities and 
other abnormal findings detected at the ARU Research Clinic will be immediately brought to the 
attention of the principal investigator and with permission from the participant, the participant’s private 
physician will be notified by letter and/or telephone depending upon the urgency of the abnormality 
detected. Participants who discontinue the study product will be urged to complete all clinic visits and 
be followed for CVD events as well as undergo the remainder of carotid artery ultrasound scans and 
other studies including those for the secondary end point. All laboratory and clinical abnormalities 
requiring referral to or follow-up by a private physician will be recorded in the participant's clinic chart. 
 
6.2  Cardiovascular Events 
 
Participants who develop angina pectoris, decreased exercise tolerance, myocardial infarction, or other 
cardiovascular events will receive care for this from a private physician. Participants will be referred to 
their private physician in accordance with the standard practice of medicine but continue on the study 
protocol unless contraindicated. Analysis of cardiovascular events will include all randomized 
participants and will be based on documentation from written medical records. Documentation of 
myocardial infarction and unstable angina must fulfill criteria outlined below. Coronary 
revascularization (CABG and PTCA) will be considered a major cardiovascular clinical event. Angina 
pectoris will be evaluated by criteria from the Rose Questionnaire [FORM 10]. All cardiovascular 
events are reported on the Study Event Record [FORM 28]. 
 
6.3  Ischemic Heart Disease Definitions 
 
6.3.1  Myocardial Infarction 
 
Symptoms: 
 
Pain or discomfort occurring in the anterior chest, back, epigastrium, jaw, neck, left shoulder, elbow, 
forearm, or wrist, which the attending physician believes to be consistent with a myocardial infarction 
(MI). These symptom criteria will not apply when there is cardiovascular collapse resulting in 
unconsciousness or if infarction occurs under general anesthesia for non-cardiac surgery. In addition, 
new onset or worsening dyspnea may be a symptom of MI if it is associated with other findings of 
myocardial necrosis (see below). 
 
Serum enzymes: 
 
The following creatine kinase (CK), creatine kinase-muscle/brain (CK-MB), troponin, aspartate amino 
transferase (AST), or lactic acid dehydrogenase (LDH) enzyme changes are defined as diagnostic of MI 
if they occur independently of conditions known to be associated with elevated levels. These conditions 
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a) In a Q-wave MI there must be: 
1) An increase of CK two times or greater than the upper limit of normal for the laboratory  
 
 
 
where the level was determined, or 
2) A positive CK-MB fraction with a 50% rise of total CK over baseline or a 50% fall in total  
 
 
 
CK relative to peak CK of any levels occurring within 48 hours of the onset of clinical  
 
 
 
symptoms, or 
3) An increase of troponin to a level considered consistent with myocardial necrosis for the  
 
 
 
laboratory where the level is determined, or 
4) Elevations of AST or LDH three times or greater than the upper limit of normal for the  
 
 
 
laboratory where the level was determined occurring within 72 hours of the onset of   
 
 
 
clinical symptoms. 
 
b) In a non-Q wave MI there must be: 
1) An increase of CK two times or greater than the upper limit of normal for the laboratory  
 
 
 
where the level was determined, or 
2) A positive CK-MB fraction with a 50% rise of total CK over baseline or a 50% fall in total  
 
 
 
CK relative to peak CK of any levels occurring within 48 hours of the onset of clinical  
 
 
 
symptoms. 
3) An increase of troponin to a level considered consistent with myocardial necrosis for the  
 
 
 
laboratory where the level is determined. 
 
c) With clinical symptoms and enzyme changes only: 
1) The CK must rise two times or greater than the upper limit of normal for the laboratory  
 
 
 
where the level is determined and the CK-MB fraction must be positive, or 
2) An increase of troponin to a level considered consistent with myocardial necrosis for the  
 
 
 
laboratory where the level is determined 
 
Electrocardiogram: 
 
a) Q-wave MI:  On a standard 12-lead ECG, new Q or QS waves with: 
a duration > 30 msec, and 
an amplitude > 0.1 mV 
demonstrated in: 
two of the three leads, II, III, or avF, or 
two adjacent leads of V1, V2, V3, V4, V5, V6, I, or avL, or growth of R waves in    
 
 
 
leads V1 or V2 with ST-T wave changes. 
 
b) Non-Q-wave MI:  On a standard 12-lead ECG, new T-wave inversions in: 
two of the three leads, II, III, avF, or 
two adjacent leads of V1, V2, V3, V4, V5, V6, I, or avL which persist for >72 hours 
Therefore, three ECGs will be required to make the diagnosis of a non-Q-wave MI: 
1)  An old ECG demonstrating no T-wave inversions, 
2)  An ECG showing new T-wave inversions, and 
 
 
 
 
3)  An ECG taken >72 hours after development of the new T-wave inversions 
 
 
 
 
 
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Criteria for diagnosis of MI: 
 
A participant must have one of the following combinations of symptoms, enzyme changes and ECG 
changes described above to meet the criteria for the diagnosis of a MI: 
 
a) Clinical symptoms, an ECG consistent with a Q-wave MI, and positive enzyme changes (for a 
 
Q-wave MI). 
b) Clinical symptoms, an ECG consistent with a non-Q-wave MI, and positive enzyme changes (for a 
 
non-Q-wave MI). 
c) With a negative ECG, clinical symptoms and positive enzyme changes (described above under 
 
serum enzymes, part c). 
d) With no symptoms and no enzyme changes, a Q-wave MI is confirmed if Q or QS waves meeting 
 
the ECG criteria above appear on serial ECGs. (In the absence of symptoms and enzyme changes, 
 
a non-Q-wave MI cannot be made). 
e) With no enzyme changes, clinical symptoms and Q or QS waves meeting the ECG criteria above 
 
for a Q-wave MI appear on serial ECGs. 
f) With no enzyme changes, clinical symptoms and T-wave inversions meeting the ECG criteria 
 
above for a non-Q-wave MI appear on serial ECGs. 
 
All non-fatal myocardial infarctions will be confirmed and recorded on the Confirmation of New Non-
fatal Myocardial Infarction Form [FORM 29] as well as reported on the Study Event Record [FORM 
28]. 
 
6.3.2  Unstable Angina/Chest Pain 
 
Transient ECG changes consisting of S-T segment elevation, S-T depression, T-wave changes, or a 
combination of these, must be documented during episodes of clinical symptoms as described above.  
These abnormalities must revert toward the control pattern after relief of pain and within 24 hours.  
There must be no ECG evidence of evolving MI (i.e., pathologic Q or QS waves). MI must be ruled out 
by serial serum enzyme studies. 
 
Hospitalization for chest pain or other clinical symptoms considered to be anginal equivalents but not 
meeting the above criteria for unstable angina will be considered chest pain. 
 
All unstable angina and chest pain events will be confirmed and recorded on the Confirmation of 
Unstable Angina/Chest Pain Form [FORM 30] as well as reported on the Study Event Record [FORM 
28]. 
 
6.4  Cerebrovascular Events 
 
Transient ischemic attacks, reversible ischemic neurologic deficits, and cerebrovascular accidents will 
be documented from written medical records and must fulfill the criteria outlined below. Subjects with 
these cerebrovascular events will continue on the study protocol unless contraindicated. Analysis of 
cerebrovascular events will include all randomized subjects. 
 
All cerebrovascular events are reported on the Study Event Record [FORM 28].

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6.5  Cerebrovascular Disease Definitions 
 
Occurrence of transient ischemic attacks, reversible ischemic neurologic deficits, and cerebrovascular 
accidents will be documented by written medical records and must fulfill the following criteria: 
 
6.5.1  Transient Ischemic Attack 
 
A sudden focal neurological deficit that completely resolves within 24 hours. 
 
All transient ischemic attack events will be confirmed and recorded on the Confirmation of Transient 
Ischemic Attack Form [FORM 31] as well as reported on the Study Event Record [FORM 28]. 
 
6.5.2  Reversible Ischemic Neurologic Deficit 
 
A focal neurological deficit that persists for more than 24 hours but in which complete neurological 
recovery occurs within 2 weeks. 
 
All reversible ischemic neurologic deficit events will be confirmed and recorded on the Confirmation of 
Reversible Ischemic Neurologic Deficit Form [FORM 32] as well as reported on the Study Event 
Record [FORM 28]. 
 
6.5.3  Cerebrovascular Accident 
 
A focal neurological deficit that permanently persists after the acute event. 
 
All cerebrovascular accident events will be confirmed and recorded on the Confirmation of 
Cerebrovascular Accident Form [FORM 33] as well as reported on the Study Event Record 
[FORM 28]. 
 
6.6  Peripheral Vascular Events 
 
Aortic aneurysms and peripheral vascular disease requiring surgical or intravascular intervention will be 
considered major events documented by written medical records. Claudication will be considered a 
symptom and evaluated by criteria defined below. Participants will continue on study protocol unless 
health care requirements for these events preclude this. Analysis of major peripheral vascular events will 
include all randomized participants. 
 
All peripheral vascular disease events requiring surgical or intravascular intervention will be confirmed 
and recorded on the Confirmation of Peripheral Vascular Disease Form [FORM 34] as well as reported 
on the Study Event Record [FORM 28]. 
 
6.7  Peripheral Vascular Disease Definitions 
 
Aortic aneurysms and peripheral vascular disease requiring surgical or revascularization intervention 
will be considered proof of disease. Participants will be considered to have claudication if they have 
reproducible pain or weakness in the calf or foot after walking a fixed distance if the pain subsides 
within a few minutes after stopping [FORM 11]. 
 
 
 

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6.8  Cancer Events 
 
The occurrence of cancer will be documented by medical records and pathology reports. Participants 
will continue in the study unless contraindicated. Analysis of cancer events will include all randomized 
participants. 
 
All cancer events will be confirmed and recorded on the Confirmation of Cancer Form [FORM 35] as 
well as reported on the Study Event Record [FORM 28]. 
 
6.9  Deep Venous Thrombotic and Pulmonary Thromboembolic Events 
 
Occurrence of deep vein thrombosis and pulmonary thromboembolism will be documented by written 
medical records and must fulfill the criteria outlined below. Participants with deep venous thrombotic 
and pulmonary thromboembolic events will continue on the study protocol unless contraindicated. 
Analysis of deep venous thrombotic and pulmonary thromboembolic events will include all randomized 
participants. 
 
6.9.1  Deep Vein Thrombosis Definition 
 
Deep vein thrombosis is frequently present in the absence of the clinical signs of pain, heat, erythema, 
and swelling, and it is absent in 50 percent of individuals in whom clinical signs and symptoms suggest 
its presence. Therefore, the diagnosis of deep vein thrombosis will be based on specific diagnostic tests. 
 These diagnostic tests include but are not limited to the following: 1) Impedance plethysmography; 2) 
Doppler flow; 3) Radiofibrinogen methods; 4) 111Indium labeled platelets; and, 5) Ascending contrast 
venography. 
 
All deep vein thrombotic events will be confirmed and recorded on the Confirmation of Deep Vein 
Thrombosis Form [FORM 36] as well as reported on the Study Event Record [FORM 28]. 
 
6.9.2  Pulmonary Thromboembolism Definition 
 
As with deep vein thrombosis, the clinical signs and symptoms of pulmonary thromboembolism are 
highly variable and usually are not solid enough to conclusively make a definitive diagnosis. Therefore, 
the diagnosis of pulmonary thromboembolism will be based on specific imaging tests, including but not 
limited to the following: 1) Pulmonary perfusion and ventilation scintiphotography; and, 2) Pulmonary 
angiography. 
 
All pulmonary thromboembolic events will be confirmed and recorded on the Confirmation of 
Pulmonary Thromboembolism Form [FORM 37] as well as reported on the Study Event Record 
[FORM 28]. 
 
6.10  Death 
 
The occurrence and cause of death will be documented by written medical records, if available, and 
death certificates. All deaths will be confirmed and recorded on the Confirmation of Death Form 
[FORM 38] as well as reported on the Study Event Record [FORM 28].

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6.11  Follow-up 
 
At annual on-trial follow-up visits (v12, v24, v36), medical history will be obtained to determine 
changes from baseline health status [FORM 39]. Any on-trial changes from baseline health status not 
previously reported will be reported on the Study Event Record [FORM 28] and other appropriate 
reporting forms described in the preceding sections. 
 
Following the last scheduled study visit, participants will be enrolled into a follow-up registry for annual 
contact and evaluation. The evaluation will include collection of historical data on major health events. 
 
Participants will complete the Closeout Form [FORM 40] at their last clinic visit. 
 
6.12  Adverse Event Reporting 
 
6.12.1  Reporting to the USC Institutional Review Board 
 
The report of a serious adverse event will be submitted, along with any other relevant information to the 
Institutional Review Board (IRB) promptly and no later than five (5) business days following the time 
that it becomes known that the subject suffered the unexpected adverse event. 
 
If an adverse event proves fatal, the IRB will be notified within 24 hours. 
 
Any unexpected adverse event that is related or possibly related to the intervention of this trial will be 
reported regardless of whether or not the adverse event is serious. The Report of the Adverse Event 
Form will be completed by the study coordinator and the adverse event discussed with the principal 
investigator who will sign prior to submission. Any unrelated adverse event or non-serious adverse 
event judged the result of progression of the disease will not be reported. 
 
6.12.2  Reporting to the External Data Safety Monitoring Board 
 
The DCC will generate a safety report enumerating all clinical events and serious and non-serious 
adverse events for the EDSMB, both on a cumulative basis (from the start of the study to present), and 
new events in the past year (see Section 9.5.2.4). Events will be presented by blinded study group. 
 
6.13  Medical Records 
 
Medical records will be requested for confirmation of all events. 
 

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7.0 
LABORATORY PROTOCOL 
 
7.1  Blood Specimen Collection 
 
Participants fast prior to each clinic visit for a minimum of eight hours (nothing by mouth except for 
water and medications). With the participant in a seated position, blood is collected by a phlebotomist 
from an antecubital vein using a tourniquet in a standardized manner. After the participant has been 
seated for 10 minutes, the tourniquet is snugly applied to the upper arm, and the blood collected into 
vacutainer tubes. Once blood flow commences, the tourniquet is immediately removed to avoid 
prolonged occlusion of the vein. Blood is collected into 10 ml and 4 ml EDTA (1.5 mg/ml wet EDTA), 
5 ml Li-heparin, 3.5 ml Na-citrate and 3.5 ml dry (with separator) vacutainer tubes depending upon the 
blood test required (Section 7.3). Blood tubes are inverted several times to thoroughly mix with the 
EDTA, Li-heparin, and Na-citrate and provided as whole blood or immediately centrifuged at 4oC or 
room temperature to separate the plasma. Serum that is collected in separator tubes sit at room 
temperature for 30 minutes prior to centrifugation. Plasma and serum are collected by low-speed 
centrifugation (1000 x g) for 10 minutes at 4oC or room temperature. All tubes are labeled with the 
participant initials, participant identification number, visit number and visit date (Section 7.2). 
 
In addition, 3.6 ml of extra plasma (2.4 ml of EDTA plasma and 1.2 ml of citrated plasma) and 1.2 ml of 
extra serum is collected at baseline and at every on-trial clinic visit, prepared as described above, and 
stored at -80oC for future studies. The plasma and serum samples are divided into 1.2 ml aliquots and 
stored at -80oC in Corning cryogenic vials with screw top caps. Plasma is stored for future 
measurements of apolipoproteins, lipoprotein particles, Lp(a), homocysteine, nitric oxide and 
coagulation factors. Serum is stored for future measurements of hormones (estradiol, estrone, estrone-
sulfate, testosterone, progesterone, sex hormone binding globulin and prolactin) and bone markers 
(bone-specific alkaline phosphatase, osteocalcin, osteoprotegrin and RANKL). The buffy coat (white 
blood cells) is collected at baseline and four times on-trial from EDTA plasma and stored at -80oC for 
future DNA studies. Urine for dipstick is collected at baseline and 1 month after randomization. 
 
7.2  Labeling, Shipment and Tracking of Samples 
 
All samples from the Atherosclerosis Research Unit Clinic are taken directly to the Atherosclerosis 
Research Core Lipid Laboratory on the same day they are collected from participants. The Core Lipid 
Laboratory serves as the central receiving point for all samples [EXHIBIT A]. Samples are sent to the 
Core Lipid Laboratory with a Laboratory Requisition Form [EXHIBIT B] indicating the laboratory 
studies to be performed as well as the samples to be stored at -80oC. 
 
Samples received by the Core Lipid Laboratory are given a laboratory sample identification number and 
logged into a central log book. In addition, the participant initials, participant identification number, date 
of sample collection, visit number, study name (NAPS) and the type of analyses to be performed are 
entered into the central log book. If a sample is to be stored at -80oC it is written in the log book as 
"stored" along with the aforementioned information. 
 
The Laboratory Requisition Form [EXHIBIT B] as well as the visit number and the table in Section 7.6 
are used by the Core Lipid Laboratory personnel to determine tests to be performed, samples to be 
stored or samples to be shipped elsewhere [EXHIBIT A]. Clinic staff resolves questions or 
discrepancies that arise concerning sample collection. 
 

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Samples to be stored are transferred quantitatively to Corning cryogenic vials with screw top caps. Each 
vial is labeled with the Core Lipid Laboratory sample identification number, participant initials, 
participant identification number, date of sample collection, visit number, study name (NAPS) and the 
vial contents (Plasma + EDTA (P+E), Plasma + CITRATE (P+C), Serum (S) and Buffy Coat). The 
buffy coat collected at v04 is labeled as described above, identified as “Buffy Coat/APOE” and stored 
for determination of apolipoprotein E genotypes. 
 
For shipment of samples to outside laboratories (Section 7.4), the same sample labeling and log 
procedure as described above is followed. Samples are hand-delivered or picked up by each respective 
laboratory. Results of laboratory tests with participant identification number, laboratory sample ID 
number, visit number and sample date is forwarded to the DCC by electronic transmission by each 
respective laboratory for data acquisition and management (Section 9.0). 

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7.3  Sample Collection Conditions 
 
LAB 
SAMPLE 
TUBE 
SIZE 
CONDITION 
FORM 
QUEST 
Chemistry Panel 
GOLD 
3.5 ml 
RT 
Serum 
Insulin 
GREEN 
5 ml 
4oC 
Plasma 
Complete Blood Count 
PURPLE 
4 ml 
RT 
Whole Blood 
Platelet Count 
Prothrombin Time (PT) 
BLUE 
3.5 ml 
RT 
Activated Partial Thromboplastin Time (aPTT) 
Fibrinogen 
HEMATOLOGY LAB 
vWF Antigen 
BLUE + 
INHIBITOR 
3.5 ml 
RT 
Whole Blood 
Tissue Plasminogen Activator (tPA) 
Plasminogen Activator Inhibitor-1 (PAI-1) 
D-Dimers 
Platelet Aggregation (ADP, Collagen, Epinephrine) 
BLUE 
3.5 ml 
RT 
Ristocetin Cofactor 
Factor VIII 
Nattokinase 
Anti-Nattokinase Antibodies 
RHEOLOGY LAB 
Erythrocyte Sedimentation Rate (ESR) 
PURPLE 
10 ml 
RT 
Whole Blood 
Red Blood Cell Aggregation (Plasma) 
Red Blood Cell Aggregability (Polymer) 
Plasma Viscosity 
Whole Blood Viscosity 
BIOCHEMISTRY LAB 
MCP-1 
PURPLE 
10 ml 
4oC 
Plasma 
IL-8 
TNFα 
IL-1β 
IL-10 
E-Selectin 
P-Selectin 
Endothelial injury in-vitro assay: VCAM-1, ICAM-1 
PURPLE 
10 ml 
RT 
Whole Blood 
Monocyte Activation (FACS): CD11b, CD11c, VLA-4 
CORE LIPID LAB 
Lipids/C-Reactive Protein (CRP) 
PURPLE 
10 ml 
4oC 
Plasma 
Hemoglobin A1c 
PURPLE 
4 ml 
RT 
Whole Blood 
Storage 
PURPLE 
10 ml 
4oC 
Plasma 
BLUE 
3.5 ml 
Plasma 
GOLD 
5 ml 
Serum 

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7.4  Specific Sample Delivery Instructions and Protocol 
 
7.4.1  Quest Laboratory 
 
A. Chemistry panel, insulin, complete blood count with platelet count, prothrombin time, 
activated partial thromboplastin time, fibrinogen 
 
1. 
 1.2 ml serum is collected in a 3.5 ml dry vacutainer tube containing a separator (gold top) at 
room temperature along with 4 ml of whole blood collected in a 4 ml EDTA (purple top) 
containing tube at room temperature. 
 
2. 
 2.5 ml plasma is collected in a 5 ml vacutainer tube containing Li-heparin (green top) at 4oC. 
 
3. 
 Along with the samples collected above, 3.5 ml, 7.0 ml or no whole blood is collected in one or 
two 3.5 ml Na-citrate (blue top) containing tubes, respectively at room temperature depending on 
the visit. 
 
4. 
The gold, green, purple and blue top tubes are labeled with participant name, date of sample 
collection, visit number, study name (NAPS) and the laboratory test to be performed: the gold 
tube is labeled “Chemistry,” the green top is labeled “Insulin,” the EDTA (purple top) containing 
tube is labeled “CBC” and one 3.5 Na-citrate (blue top) containing tube is labeled “PT/aPTT” 
and one 3.5 Na-citrate (blue top) containing tube is labeled “Fibrinogen.” 
 
5. 
The Quest Laboratory Requisition forms are completed for each appropriate laboratory test. 
 
6. 
The collection tubes and the Quest Laboratory Requisition forms are delivered to the Core Lipid 
Laboratory, assigned sample ID numbers, logged into the system and the Quest courier is 
notified for same day pick up of samples. 
 
B. Delivery of samples to Quest Laboratory 
 
Contact information: 
 
  
  
 
Telephone: (818) 737-8347 
 
7.4.2  Hematology Laboratory – Dr. Howard Liebman 
 
A. Coagulation Factors (vWF antigen, tissue plasminogen activator, plasminogen activator 
inhibitor-1, D-dimers) and Heme Factors (platelet aggregation (ADP, collagen, epinephrine), 
ristocetin cofactor, factor VIII, plasma nattokinase levels and anti-nattokinase antibodies) 
 
1. 
14 ml of whole blood is collected in one 3.5 Na-citrate (blue top) + inhibitors containing tube 
(prepared by Hematology Laboratory for coagulation factors) and three 3.5 ml Na-citrate (blue 
top) containing tubes at room temperature. 
 
2. 
The four 3.5 ml blue top tubes are labeled with participant initials, participant identification 
number, date of sample collection, visit number, study name (NAPS) and the panel analytes to be 
performed; the one 3.5 Na-citrate (blue top) + inhibitors tube is labeled “Coagulation” and the 
three 3.5 ml Na-citrate (blue top) containing tubes are labeled “Heme/+Platelet.” 
 

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3. 
The four 3.5 ml blue top tubes are immediately delivered to the Hematology Laboratory under 
the Directorship of Dr. Howard Liebman. 
 
B. Coagulation Factors and Heme Factors without Platelet Aggregation Studies 
 
1. 
For the visits that do not require the platelet aggregation studies, 10.5 ml of whole blood is 
collected in one 3.5 Na-citrate (blue top) + inhibitors containing tubes (prepared by Hematology 
Laboratory) and two 3.5 ml Na-citrate (blue top) containing tubes at room temperature. 
 
2. 
The three 3.5 ml blue top tubes are labeled with participant initials, participant identification 
number, date of sample collection, visit number, study name (NAPS) and the panel analytes to be 
performed; the one 3.5 Na-citrate (blue top) + inhibitors tube is labeled “Coagulation” and the 
two 3.5 ml Na-citrate (blue top) containing tubes are labeled “Heme/–Platelet.” 
 
3. 
The three 3.5 ml blue top tubes are immediately delivered to the Hematology Laboratory under 
the Directorship of Dr. Howard Liebman. 
 
C. Nattokinase Plasma Levels (when not collected as part of the Coagulation and Heme Factors) 
 
1. 
3.5 ml of whole blood is collected in a 3.5 ml Na-citrate (blue top) containing tube at room 
temperature. 
 
2. 
The 3.5 ml blue top tube is labeled with participant initials, participant identification number, 
date of sample collection, visit number, study name (NAPS) and labeled with the type of analysis 
to be performed “Nattokinase.” 
 
3. 
The 3.5 ml blue top tube is immediately delivered to the Hematology Laboratory under the 
Directorship of Dr. Howard Liebman. 
 
D.  Delivery of Samples to Hematology Laboratory 
 
Laboratory address: 
 
 
Howard A. Liebman, M.D. 
 
USC/Norris Comprehensive Cancer Center and Hospital 
 
1441 Eastlake Avenue 
 
Topping Tower Building, Room NOR 3466 
 
Los Angeles, CA 90033 
 
 
Telephone: (323) 865-3950 
 
Mobile: (323) 980-0820 
 
Email: Liebman_h@med.usc.edu 
 
Contact personnel: 
 
Leanne Rochanda 
 
Keck School of Medicine 
1441 Eastlake Avenue 
Ezralow Tower Building, Room NOR 5338A 
Los Angeles, CA 90033 

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Telephone: (323) 865-0965  
 
             
Mobile: (925) 708 1471 
Email: rochanda@usc.edu 
 
7.4.3 Rheology Laboratory – Dr. Herbert Meiselman 
 
A.  Rheology Measures (erythrocyte sedimentation rate, red blood cell aggregation, red blood cell 
  aggregability, plasma viscosity, whole blood viscosity) 
 
1. 
10 ml of whole blood is collected in a 10 ml EDTA (purple top) containing tube at room 
temperature. 
 
2. 
The 10 ml purple top tube is labeled with participant initials, participant identification number, 
date of sample collection, visit number, study name (NAPS) and labeled with the panel of 
analytes to be performed “Viscosity.” 
 
3. 
The 10 ml purple top tube is immediately delivered to the Rheology Laboratory under the 
Directorship of Dr. Herbert Meiselman. 
 
B.  Delivery of samples to Rheology Laboratory 
 
Laboratory address: 
 
 
 
 
Herbert J. Meiselman, Sc.D. 
 
 
 
Keck School of Medicine 
 
 
 
1333 San Pablo Street 
 
 
 
Mudd Building, Room 120 
 
 
 
Los Angeles, CA 90033 
 
 
 
 
Telephone: (323) 442-1268 or (323) 442-1267 
 
 
 
Email: hmeiselman@gmail.com or meiselman@usc.edu 
 
Contact personnel: 
 
 
 
 
Rose Wenby 
 
 
 
Keck School of Medicine 
 
 
 
1333 San Pablo Street 
 
 
 
Mudd Building, Room 120 
 
 
 
Los Angeles, CA 90033 
 
 
 
 
Telephone: (323) 442-1268 or (323) 442-1267 
 
 
 
Email: rwenby@usc.edu 
 
 
 
 
 
 
 

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30 
7.4.4  Biochemistry Laboratory – Dr. Vijay Kalra 
 
A.  Inflammatory Markers (MCP-1, IL-8, TNFα, IL-1β, IL-10, E-selectin, P-selectin) 
 
At screening visit: 
 
1. 
5 ml of plasma is collected in a 10 ml EDTA (purple top) containing tube at 4°C. 
 
2. 
The plasma sample (5 ml) is divided into 2 aliquots. The first aliquot of 2.5 ml is transferred to a 
3.0 ml screw-capped plastic cryogenic tube that can withstand the temperature of -80°C. The 
tube is labeled "Inflammatory." The second aliquot of 2.5 ml is used to analyze lipid levels and 
C-Reactive Protein (CRP) in the Core Lipid Laboratory. 
 
At on-trial visits: 
 
1. 
5 ml of plasma is collected in a 10 ml EDTA (purple top) containing tube at 4°C. 
 
2. 
The plasma sample (5 ml) is divided into 2 equal aliquots and a buffy coat collected from the 
EDTA sample. The first aliquot of 2.5 ml is transferred to a 3.0 ml screw-capped plastic 
cryogenic tube that can withstand the temperature of -80°C. The tube is labeled "Inflammatory." 
The second aliquot of 2.5 ml is divided equally and transferred to two 2.0 ml screw-capped 
plastic cryogenic tubes that can withstand the temperature of -80°C. The tubes are labeled 
"EDTA Plasma” and stored in the Core Lipid Laboratory. The buffy coat is collected from the 
EDTA plasma sample and transferred to a 2.0 ml screw-capped plastic cryogenic tube that can 
withstand the temperature of -80°C. The tube is labeled "Buffy Coat” and stored in the Core 
Lipid Laboratory. 
 
At screening visit and on-trial visits: 
 
3. 
The screw-capped plastic cryogenic tubes are labeled with participant initials, participant 
identification number, date of sample collection, visit number, study name (NAPS) and the panel 
of analytes to be performed (Inflammatory). Water-insoluble ink is used and the labels protected 
by wrapping them with transparent tape. 
 
4. 
The screw-capped plastic cryogenic tubes are immediately frozen at -80oC until delivery to the 
Biochemistry Laboratory under the Directorship of Dr. Vijay Kalra. 
 
B. In-Vitro Studies: Endothelial Injury In-Vitro Assay: VCAM-1, ICAM-1 and Monocyte 
Activation (FACS): CD11b, CD11c, VLA-4 
 
1. 
10 ml of whole blood is collected in a 10 ml EDTA (purple top) containing tube at room 
temperature. 
 
2. 
The 10 ml purple top tube is labeled with participant initials, participant identification number, 
date of sample collection, visit number, study name (NAPS) and labeled with the type of analysis 
to be performed “In-Vitro.” 
 
3. 
The 10 ml purple top tube is immediately shipped to the Biochemistry Laboratory under the 
Directorship of Dr. Vijay Kalra. 

Manual of Operations 
 
 
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31 
C.  Delivery of Samples to Biochemistry Laboratory 
 
Laboratory address: 
 
 
 
 
Vijay Kalra, Ph.D. 
 
 
Keck School of Medicine 
 
 
2011 Zonal Avenue 
Hoffman Medical Research Building (HMR), Room 611 / 613 
Los Angeles, CA 90033 
 
Telephone: (323) 442-1526 
Laboratory: (323) 442-1528 
Mobile: (626) 388-5924 
Email: vkalra@usc.edu 
 
Contact personnel: 
 
 
 
 
Caryn Gonsalves, Ph.D. 
 
 
Keck School of Medicine 
 
 
2011 Zonal Avenue 
Hoffman Medical Research Building (HMR), Room 611 / 613 
Los Angeles, CA 90033 
 
Telephone: (323) 442-1528 
Mobile: (626) 353-8232 
 

Manual of Operations 
 
 
Version: 03-10-14 
 
32 
7.5  Schematic of Blood Sample Flow 
 
 
Chemistry panel
Insulin
Complete blood count (CBC)
Platelet count
Prothrombin time (PT)
Activated partial thromboplastin time (aPTT)
Fibrinogen
Lipids
C-Reactive Protein (CRP)
Hemoglobin A1c
Apolipoprotein E genotype
Storage:   Plasma (EDTA)
Plasma (citrate)
Serum
Buffy coat
MCP-1
IL-8
TNFα
IL-1β
IL-10
E-selectin
P-selectin
Endothelial injury in-vitro assay: VCAM-1
ICAM-1
Monocyte activation (FACS): CD11b
CD11c
VLA-4
vWF antigen
Tissue plasminogen activator (tPA)
Plasminogen activator inhibitor-1 (PAI-1)
D-dimers
Platelet aggregation
Ristocetin factor
Factor VIII
Nattokinase levels
Anti-nattokinase antibodies
Erythrocyte sedimentation rate (ESR)
Red blood cell aggregation (plasma)
Red blood cell aggregability (polymer)
Plasma viscosity
Whole blood viscosity
ARU CLINIC: Blood drawn
Courier to Quest
Core Lipid Laboratory 
Hematology Laboratory
Biochemistry Laboratory
Rheology Laboratory

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7.6  Sample Flow Schedule of Biochemical Safety and Laboratory Tests 
 
LAB 
 
RESEARCH ACTIVITY 
SCREEN 
BASELINE 
WEEK 1 
01 
02 
03 
04 
05 
06 
09 
12 
15 
18 
21 
24 
27 
30 
33 
36 
QUEST 
Chemistry Panel1 
x 
  
  
x 
x 
x 
x 
x 
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Insulin2 
x 
  
  
  
  
x 
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Complete Blood Count3 
x 
  
  
x 
x 
x 
x 
x 
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Platelet Count3 
x 
  
  
x 
x 
x 
x 
x 
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Prothrombin Time (PT)4 
x 
  
  
x 
  
x 
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Activated Partial Thromboplastin Time 
(aPTT)4 
x 
  
  
x 
  
x 
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Fibrinogen4 
  
x 
x 
x 
  
  
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
HEMATOLOGY LAB 
vWF Antigen5 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Tissue Plasminogen Activator (tPA)5 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Plasminogen Activator Inhibitor-1 (PAI-1)5 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
D-Dimers5 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Platelet Aggregation (ADT, Collagen, 
Epinephrine)4 
  
x 
  
x 
  
  
  
  
x 
  
  
  
  
  
  
  
  
  
  
Ristocetin Cofactor4 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Factor VIII4 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Nattokinase4 
  
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Anti-Nattokinase Antibodies4 
  
x 
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
RHEOLOGY LAB 
Erythrocyte Sedimentation Rate (ESR)3 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Red Blood Cell Aggregation (Plasma)3 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Red Blood Cell Aggregability (Polymer)3 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Plasma Viscosity3 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
Whole Blood Viscosity3 
  
x 
x 
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
  
BIOCHEMISTRY LAB 
MCP-16 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
IL-86 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
TNFα6 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
IL-1β6 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
IL-106 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
E-Selectin6 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
P-Selectin6 
x 
  
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
Endothelial injury in-vitro assay: VCAM-1, 
ICAM-13 
  
x 
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
Monocyte Activation (FACS): CD11b, 
CD11c, VLA-43 
  
x 
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
CORE LIPID LAB 
Lipids/C-Reactive Protein (CRP)6 
x 
  
  
  
  
  
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Hemoglobin A1c3 
x 
  
  
  
  
x 
  
  
x 
  
x 
  
x 
  
x 
  
x 
  
x 
Storage1,6,7 
  
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Buffy Coat 
  
x 
  
  
x 
  
  
  
  
x 
  
  
  
x 
  
  
  
x 
  
ApoE Genotype 
  
  
  
  
  
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
Urine Dipstick 
  
x 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 

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1 Collect serum in dry gold top tubes with separator 
2 Collect plasma in Li-heparin (green top) tube 
3 Collect whole blood in EDTA (purple top) tube 
4 Collect whole blood in Na-citrate (blue top) tube 
5 Collect whole blood in special Na-citrate (blue top) tube with inhibitors supplied by Heme Lab 
6 Collect plasma in EDTA (purple top) tube 
7 Collect plasma in in Na-citrate (blue top) tube 
 
 

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35 
7.7  ARU Clinic Sample Flow  
 
Samples conducted by the ARU clinic 
 
1. Urine is collected for urine dipstick. 
 
Samples delivered to Core Lipid Laboratory from the ARU clinic [EXHIBIT A] 
 
A.  Samples prepared by ARU clinic for delivery Quest Laboratory 
 
1. Serum for chemistry panel, plasma for insulin and whole blood for CBC and platelet count and 
whole blood for prothrombin (PT) and activated partial thromboplastin time (aPTT) and fibrinogen 
are labeled by ARU personnel with participant name, visit number, date, type of analysis to be 
performed and study name (NAPS). 
 
2. The Quest laboratory requisition form is completed by ARU personnel and accompanies the 
samples. 
 
B.  Samples to be internally processed 
 
Whole Blood Samples 
 
1. Whole blood is collected in "bulk" and the collection tubes appropriately labeled by ARU personnel 
indicating to the Core Lipid Laboratory personnel what panel of analytes are to be determined. In 
addition, the labels contain participant initials, participant identification number, visit number, date 
and study name (NAPS). 
 
2. The Laboratory Requisition Form [EXHIBIT B] accompanies the whole blood samples indicating to 
the Core Lipid Laboratory personnel which panel of analytes are to be determined (Section 7.5). 
 
Plasma and Serum Samples 
 
1. Plasma is collected in "bulk" and the tubes appropriately labeled by ARU personnel indicating to the 
Core Lipid Laboratory personnel that the sample is plasma + EDTA (Plasma) and plasma + citrate 
(Citrated Plasma). In addition, labels contain participant initials, participant identification number, 
visit number, date and study name (NAPS). Plasma + EDTA samples are prepared in a cold 
environment (4oC) or room temperature; plasma + citrate samples are prepared at room temperature. 
 
2. The Laboratory Requisition Form [EXHIBIT B] accompanies the plasma samples indicating to the 
Core Lipid Laboratory personnel which immediate determinations are to be performed with the 
plasma and indicating that the remaining sample is plasma storage (Section 7.5). 
 
3. Serum is collected in "bulk" and the tubes appropriately labeled by ARU personnel indicating to the 
Core Lipid Laboratory personnel that the sample is SERUM. In addition, the labels contain 
participant initials, participant identification number, visit number, date and study name (NAPS). 
The serum sample is prepared at room temperature. 
 
4. The Laboratory Requisition Form [EXHIBIT B] accompanies the serum samples indicating to the 
Core Lipid Laboratory personnel that the sample is serum storage (see Section 7.5). 
 

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36 
7.8  Core Lipid Laboratory Sample Flow [EXHIBIT A] 
 
1. Samples received from the ARU clinic are logged in and assigned a laboratory ID number by the 
 
Core Lipid Laboratory personnel as described in Section 7.2. 
 
2. Serum for chemistry panel, plasma for insulin and whole blood for CBC and platelet count and 
whole blood for prothrombin (PT) and activated partial thromboplastin time (aPTT) and fibrinogen 
are accompanied by the Quest laboratory requisition form completed by ARU personnel. The 
samples are logged in as above, packaged in plastic bags (samples and Quest requisition form for 
each participant) and placed in the Quest pick up box. Samples are delivered to the Quest laboratory 
on a daily basis by a Quest messenger. 
 
3. Collection tubes containing whole blood are accompanied with the Laboratory Requisition Form 
[EXHIBIT B] indicating to the Core Lipid Laboratory personnel what panel of analytes are to be 
determined and to which laboratory samples are to be shipped. The Core Lipid Laboratory personnel 
deliver samples to the appropriate laboratories (see Section 7.5). 
 
4. Type of determinations to be performed on "bulk" samples and whether samples are for storage 
(plasma + EDTA, plasma + citrate, serum) is indicated on the Laboratory Requisition Form 
[EXHIBIT B]. 
 
5. "Bulk" plasma samples drawn for lipids/CRP (plasma + EDTA) are aliquoted, labeled with 
participant initials, participant identification number, laboratory ID number, date, visit number, study 
name (NAPS) and labeled with type of analysis to be performed by the Core Lipid Laboratory 
personnel “Lipids/CRP.” Lipid and CRP determinations are performed by Core Lipid Laboratory 
personnel. 
 
6. Collection tubes (plasma + EDTA) containing whole blood drawn for hemoglobin A1c are labeled 
with participant initials, participant identification number, laboratory ID number, date, visit number, 
study name (NAPS) and labeled with type of analysis to be performed by the Core Lipid Laboratory 
personnel “Hemoglobin A1c.” Hemoglobin A1c determinations are performed by Core Lipid 
Laboratory personnel. 
 
7. The buffy coat is prepared by Core Lipid Laboratory personnel from EDTA plasma and transferred 
into a cryogenic tube, labeled with participant initials, participant identification number, visit 
number, date, study name (NAPS) and the words "Buffy Coat" and stored at -80oC as outlined in the 
next section. 
 
8. The buffy coat collected at v04 is identified for apolipoprotein E genotype and stored at -80oC as 
outlined in the next section. 
 
9. Plasma + EDTA, plasma + citrate and serum identified for storage at -80oC follow the protocol 
outlined in the next section. 
 
7.9  Sample Storage 
 
1. The following -80oC sample storage is proposed: 1) 2 cryotubes of 1.2 ml plasma + EDTA; 2) 1 
cryotube of 1.2 ml plasma + citrate; 3) 1 cryotube of 1.2 ml serum; and, 4) buffy coats. 
2. Plasma and serum storage samples are collected at baseline and at every clinic visit for a total of 18 
collections each. 

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37 
3. Buffy coats are collected at the baseline visit and at on-trial clinic visits with preparation of “bulk” 
plasma for sample determination of inflammatory markers and storage for a total of 5 collections. 
 
4. Plasma + EDTA received by the Core Lipid Laboratory for storage is placed into 2 cryogenic tubes 
with 2 ml capacity in 1.2 ml aliquots. The cryogenic tubes are labeled by the Core Lipid Laboratory 
personnel with a cryogenic label containing participant initials, participant identification number, 
laboratory ID number, date, visit number, study name (NAPS) and type of sample “EDTA Plasma.” 
 
5. Plasma + citrate received by the Core Lipid Laboratory for storage is placed into 1 cryogenic tube 
with 2 ml capacity in a 1.2 ml aliquot. The cryogenic tube is labeled by the Core Lipid Laboratory 
personnel with a cryogenic label containing participant initials, participant identification number, 
laboratory ID number, date, visit number, study name (NAPS) and type of sample “EDTA Plasma.” 
 
6. Serum received by the Core Lipid Laboratory for storage is placed into 1 cryogenic tube with 2 ml 
capacity in a 1.2 ml aliquot. The cryogenic tube is labeled by the Core Lipid Laboratory personnel 
with a cryogenic label containing participant initials, participant identification number, laboratory ID 
number, date, visit number, study name (NAPS) and type of sample “Serum.” 
 
7. The buffy coat is labeled with participant initials, participant identification number, visit number, 
date, study name (NAPS) and labeled "Buffy Coat" and stored at -80oC. 
 
8. The buffy coat collected at v04 is identified for apolipoprotein E genotype, labeled with participant 
initials, participant identification number, laboratory ID number, date, visit number, study name 
(NAPS) and type of analysis to be performed (Buffy Coat/APOE). The sample is stored at -80oC and 
delivered to Dr. Hooman Allayee’s laboratory for determination of apolipoprotein E genotypes. 
 
7.10  Quality Control Procedures 
 
The Core Lipid Laboratory (Dr. Hwang) is a CLIA certified laboratory. The Core Lipid Laboratory is 
also enrolled in the Centers for Disease Control (CDC) lipid standardization program. The CDC sends 
reference plasma samples each quarter to the laboratory that is then analyzed for total cholesterol, total 
triglyceride and high density lipoprotein levels, the results of which are sent back to the CDC. The CDC 
then notifies the laboratory of the performance relative to the standard. A record of the results for each 
analyte is kept. For special laboratory determinations analyses of variance and standards have been 
established. Procedures for following variability and precision of laboratory determinations are given in 
Section 9.0. 
 
7.11  Laboratory Determinations 
 
7.11.1  Chemistry Panel 
 
  1) albumin 
 
 
12) GGTP 
  2) albumin/globulin ratio  
13) glucose 
  3) alkaline phosphatase 
 
14) iron 
  4) ALT (SGPT) 
 
 
15) LDH 
  5) AST (SGOT) 
 
 
16) phosphorous 
  6) bicarbonate 
 
 
17) potassium 
  7) BUN 
 
 
18) sodium 
  8) BUN/creatinine ratio 
 
19) total bilirubin 
  9) calcium 
 
 
20) total protein 

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38 
10) chloride 
 
 
21) uric acid 
11) creatinine 
 
Blood chemistries are determined by standard analytical methods by Quest in Los Angeles California.  
Chemistry results are sent to the ARU clinic; clinic staff will send a photocopy of the results to the DCC 
for data entry. 
 
7.11.2  Insulin 
 
Plasma insulin levels are determined by standard analytical methods by Quest in Los Angeles 
California. Plasma insulin results are sent to the ARU clinic; clinic staff will send a photocopy of the 
results to the DCC for data entry. 
 
7.11.3  Complete Blood Count and Platelet Count 
 
  1) WBC 
  2) RBC 
  3) hemoglobin 
  4) hematocrit 
  5) MCV 
  6) MCH 
  7) MCHC 
  8) RDW 
  9) platelet count 
10) manual differential 
 
Complete blood count along with platelet count is determined by standard analytical methods by Quest 
in Los Angeles California. Complete blood count results are sent to the ARU clinic; clinic staff will send 
a photocopy of the results to the DCC for data entry. 
 
7.11.4  Prothrombin time (PT) and Activated Partial Thromboplastin Time (aPTT) 
 
PT and aPTT are determined by standard analytical methods by Quest in Los Angeles California. PT 
and aPTT results are sent to the ARU clinic; clinic staff will send a photocopy of the results to the DCC 
for data entry. 
 
7.11.5  Fibrinogen 
 
Fibrinogen is determined by standard analytical methods by Quest in Los Angeles California. 
Fibrinogen results are sent to the ARU clinic; clinic staff will send a photocopy of the results to the DCC 
for data entry. 
 
7.11.6  vWF Antigen 
 
VWF antigen is measured by ELISA with a pooled normal plasma standard curve. Results are reported 
as a percentage of the normal pool (1,2). 
 
7.11.7  Tissue Plasminogen Activator (tPA) 
 
tPA in plasma is measured by ELISA (3). 

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7.11.8  Plasminogen Activator Inhitor-1 (PAI-1) 
 
PA-1 in plasma is measured by ELISA (3). 
 
7.11.9  D-Dimers 
 
D-dimers in plasma are measured by ELISA (3,4). 
 
7.11.10  Platelet Aggregation (ADP, Collagen, Epinephrine) 
 
Platelet aggregation is determined with a Chrono-Log Model 700 Whole Blood/Optical Lumi 
Aggregometer (Havertown, PA) that measures platelet function in samples using electrical impedance in 
whole blood or optical density in plasma. Platelet aggregation is measured in the presence of ADP, 
collagen and to epinephrine (5). 
 
7.11.11  Ristocetin Cofactor 
 
Platelet aggregation with Ristocetin Cofactor (von Willenbrand Factor Assay) is determined in plasma 
with a Chrono-Log Model 700 Whole Blood/Optical Lumi Aggregometer (Havertown, PA). 
 
7.11.12  Factor VIII 
 
Factor VIII activity is measured by a modified aPTT assay utilizing factor VIII deficient plasma and 
serial dilutions of participant plasma with a pooled normal plasma standard curve. Values are reported as 
a percentage of normal plasma (1,2). 
 
7.11.13  Nattokinase and Anti-Nattokinase Antibodies 
 
Nattokinase levels and anti-nattokinase antibodies are measured in plasma by ELISA. 
 
7.11.14  Erythrocyte Sedimentation Rate (ESR) 
 
RBC aggregation is evaluated by the ESR method in which Westergren tubes (i.e., 3 mm ID by 200 mm 
high) are filled with blood and positioned vertically for one hour: the distance of RBC sedimentation 
reflects RBC aggregation (6). 
 
7.11.15  Red Blood Cell Aggregation 
 
In plasma or appropriate polymer solutions, RBCs undergo reversible aggregation into face-to-face 
linear or three-dimensional structures with the extent of aggregation in plasma reflecting the 
concentration of large proteins (e.g., fibrinogen). A Myrenne RBC Aggregometer (Myrenne GmbH, 
Roetgen, Germany) is used to determine the extent of aggregation at stasis and at very low shear for 
cells in autologous plasma and in a pro-aggregating polymer solution (e.g., 3% 70 kDa dextran in 
isotonic PBS). The instrument has a cone-plate shearing geometry with the blood sample placed in the 
gap between these surfaces. An IR source and detector measure the light transmission through the 
sample; greater aggregation yields increased light transmission. The aggregometer is computer-
controlled with results displayed on a monitor (6,7). 
 
 
 

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7.11.16  Red Blood cell Aggregability 
 
The cellular factors that influence RBC aggregation are termed “aggregability” to indicate that they refer 
to the cell and not the suspending medium, is evaluated using the Myrenne Aggregometer. In this 
procedure, RBCs are washed twice in isotonic PBS (pH=7.4, 290 mOsm/kg) then once in a defined pro-
aggregating medium of dextran, following which the RBC-dextran suspension is adjusted to a 
hematocrit of 0.4 L/L. A 3% solution of 70 kDa dextran in PBS is selected for these studies since it 
induces moderate aggregation and represents a stable, well-defined medium. Comparisons of different 
RBC populations (e.g., time-based samples from a subject) will indicate any alterations of aggregability 
(6,8). 
 
7.11.17  Plasma Viscosity 
 
Plasma viscosity is measured using a small volume Couette viscometer (Contraves LS-30, Contraves 
AG, Zurich, Switzerland) at 37ºC. An appropriate volume of blood is centrifuged at 2,000 x g and the 
plasma carefully removed and saved. Viscosity testing is done either on the day the blood sample is 
received or stored at -80 ºC for a brief period. Results are reported as mPa.s, which is numerically equal 
to centipoise (9). 
 
7.11.18  Whole Blood Viscosity 
 
The viscosity of EDTA-treated whole blood is measured using an automated scanning capillary 
viscometer (Rheolog, Rheovector, Co.) at 37ºC over a shear rate range of 1 to 1,000 s-1. About 2.5 ml of 
blood are needed per measurement. The device has a horizontal small-diameter glass capillary with 
vertical tubes at each end and photo detectors to sense the height of blood in each vertical tube. Initially, 
blood is introduced into the system so that these heights are unequal then the system is isolated and 
blood flows through the capillary due to the difference in heights; flow and pressure gradient (i.e., 
heights in both tubes) are constantly measured during flow. The resulting data are analyzed using 
vendor-supplied software; the results, as viscosity at each shear rate, are displayed and available for 
printing (6,9). 
 
7.11.19  Inflammatory Markers 
 
 
1) MCP-1 
 
 
2) IL-8 
 
 
3) TNFα 
 
 
4) IL-1β 
 
 
5) IL-10 
 
MCP-1, IL-8, TNFα, IL-1β and IL-10 are measured in plasma using a multiplex ELISA kit by 
procedures previously established in our laboratory (10,11). 
 
7.11.20  E-Selectin 
 
Plasma levels of E-selectin and VCAM-1 (as a control) are measured by ELISA as previously described 
(12). 
 
7.11.21  P-Selectin 
 
Plasma levels of P-selectin are measured by ELISA as previously described (12). 

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7.11.22  Endothelial injury In-vitro Assay 
 
 
1) VCAM-1 
 
2) ICAM-1 
 
Expression of endothelial adhesion molecules (VCAM-1 and ICAM-1) by circulating monocytes is 
performed in cultured human aortic endothelial cells using isolated monocytes (13) 
 
7.11.23  Monocyte Activation (FACS) 
 
 
 
1) CD11b 
 
 
2) CD11c 
 
 
3) VLA-4 
 
Activation of monocytes are determined as the expression of the monocyte cell surface markers 
CD11b/CD11c and VLA-4 using fluorescence-activated cell sorting (FACS) of monocytes isolated from 
whole blood (14) 
 
7.11.24  Lipids 
 
 
1) calculated LDL-C 
 
2) plasma total cholesterol 
 
3) plasma total triglycerides 
 
4) plasma HDL-C 
 
5) estimated VLDL-C 
 
Plasma total cholesterol (TC), triglyceride (TG), and HDL-cholesterol levels are determined by 
enzymatic assays and standardized to the CDC using the Lipid Research Clinic protocol (15). 
HDL-cholesterol is measured after precipitation of the apoprotein B-containing lipoproteins (LDL and 
VLDL) in whole plasma by heparin-manganese chloride (16). Very low density lipoprotein cholesterol 
(VLDL-C) is assumed to equal one-fifth of the plasma triglyceride level and LDL-C is calculated using 
the Freidewald equation (17): 
 
 
LDL-C=TC-(TG/5+HDL-C) 
 
If the triglyceride level exceeds 500 mg/dL, direct measurement of LDL-C is performed. 
 
After accumulation of 20 to 24 samples, a work sheet is prepared for the samples to be analyzed for the 
above lipid values. The results printed from the VPSS are transferred to the worksheet and entered into a 
computerized Excel database and transmitted to the DCC both electronically and by hard copy for 
double entry key stroke verification. 
 
7.11.25  C-Reactive Protein (CRP) 
 
CRP is measured in plasma by a turbidimetric assay by measuring the CRP-phospholipid complex at an 
absorbance of 660/700 nm. 
 
 
 
 

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7.11.26  Hemoglobin A1c 
 
Hemoglobin A1c is measured by turbidimetric inhibition immunoassay in hemolyzed whole blood at an 
absorbance of 340/659 nm. 
 
7.11.27  Peripheral Blood Buffy Coat Preparation 
 
General: 
 
When a tube of anticoagulated whole blood is centrifuged at 2,200 rpm for 8 minutes in a centrifuge, it 
separates into three main layers: 
 
1)  Plasma 
2)  Buffy coat layer containing white blood cells, nucleated red blood cells and platelets 
3)  erythrocyte layer 
 
Specimen Requirement: 
 
EDTA anticoagulated whole blood 
 
Equipment: 
 
1) 7 ml or 10 ml EDTA tube 
2) Wintrobe tube or equivalent 
3) 9 inch Pasteur pipettes 
4) Rubber bulbs 
5) 15 ml red top tube to hold Wintrobe tube for centrifugation 
6) Centrifuge 
 
Procedure: 
 
1) Centrifuge the EDTA (purple top) tube at 2,200 rpm for 8 minutes. If the amount of specimen is 2 
 
ml or less, transfer into Wintrobe tube(s) and proceed to step #4 below. 
 
2) Using a 9 inch Pasteur pipette, discard about two-thirds of the plasma. 
 
3) Aspirate the remaining plasma, the buffy coat layer, and the packed red cells just below the buffy 
coat layer. Transfer the mixture to a Wintrobe tube, using as many tubes as necessary. 
 
4) Place the Wintrobe tube into a 15 ml red top tube and seal with the top. Centrifuge the tube for 8 
 
minutes at 2,200 rpm. 
 
5) Aspirate the plasma, the buffy coat layer and about 1 mm of packed red blood cells directly beneath 
 
the buffy coat. 
 
6) Transfer the mixture to a cryogenic tube placing the mixture directly on the bottom of the tube 
 
without dripping it down the sides of the tube. Label the tube (Section 7.2). 
 
 
 

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7.11.28  ApoE Genotype 
 
Determination of the apoE2/E3/E4 alleles uses SNP genotypes derived from two SNPs, rs429358 and 
rs7412, as reported previously (18). Genotyping is performed using the Applied Biosystems, Inc. (ABI) 
TaqMan system. Briefly, for each SNP, a PCR reaction containing 2 ng of genomic DNA, amplification 
primers and two 20-30 bp oligonucleotides encompassing the polymorphic site is carried out according 
to the manufacturer's protocols. 
 
7.12  Clinical Determinations 
 
7.12.1  Electrocardiogram 
 
Standard 12-lead ECG is performed by trained ARU research clinic personnel by standard technique. 
 
7.12.2  Waist:Hip Ratio 
 
The waist circumference is measured and recorded [FORM 23] in the standard manner (19) with the 
participant standing erect, the abdomen relaxed, the feet together and the arms to the side. An inelastic 
tape measure is placed directly on the skin around the subject at the narrowest part of the torso between 
the ribs and iliac crest as seen from the anterior aspect. The measurement is taken at the end of a normal 
expiration with the tape measure in a horizontal plane without compression of the skin. With tape 
readings to the nearest 0.1 cm as described above, the waist circumference measurement of a participant 
is accurate within +1 cm of the "true" waist circumference. 
 
The hip (or buttocks) circumference is measured in the standard manner (19) with the participant 
standing erect and wearing nonrestrictive underwear. The level of maximum extension of the buttocks is 
identified and an inelastic tape measure is placed around this region in a horizontal plane without 
compressing the skin. Under these conditions and with tape readings to the nearest 0.1 cm, the hip 
measurement is accurate within +1 cm of the "true" hip circumference. The waist:hip ratio is an 
indicator of the pattern of subcutaneous adipose tissue distribution that has been shown to correlate with 
the risk of diabetes mellitus and CHD (20,21). 
 
7.12.3  Body Weight 
 
Height and weight is measured (in street clothes without shoes) at v00 and weight is measured at each 
subsequent clinic visit for the duration of the trial. 
 
7.13  References 
 
  1. Liebman HA, Comenzo R, Allen S, DiIorio J. A glycosaminoglycan inhibitor of thrombin: a new 
mechanism for abnormal hemostatic assays in cancer. Am J Hematol 1991;38:24-29. 
  2. Rochanda L, del Zoppo GJ, Feinstein DI, Liebman HA. Approach to treatment, characterization, 
and diagnosis of an acquired auto-antibody directed against prothrombin, factor X and factor IX: a case 
report and review of the literature. Haemophilia 2011;18:102-107. 
  3. Liebman HA, Carfagno MK, Weitz I, Berard P, DiIorio J, Vosburgh E, Simms R. Excessive 
fibrinolysis in amyloidosis resulting from elevated plasma single chain urokinase. Am J Clin Pathol 
1992;98:534-541. 
  4. Weitz IC, Razavi P, Ghods M, Rochanda L, Zwicker J, Furie B, Manly D, Mackman N, Liebman 
HA. Eculizumab therapy results in rapid and sustained decreases in markers of thrombin generation and 
inflammation in patients with paroxysmal nocturnal hemoglobinuria. Thromb Res 2012;130:361-368. 

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44 
  5. Alexy T, Tucker S, Boyle S, Rowe VL, Weaver FA, Liebman HA. Heparin-PF4 antibodies are 
frequent following vascular surgery, but are not a frequent cause of graft thrombosis or 
thrombocytopenia. J Vasc Surg 2008;48:377-381. 
  6. Baskurt OK, Uyuklu M, Ulker P, Cengiz M, Nemeth N, Alexy T, Shin S, Hardeman MR, 
Meiselman HJ. Comparison of three instruments for measuring red blood cell aggregation. Clin 
Hemorheol Microcirc 2009;43:283-289. 
  7. Lee BK, Alexy T, Wenby RB, Meiselman HJ. Red blood cell aggregation quantitated via Myrenne 
Aggregometer and yield shear stress. Biorheology 2007;44:29-36. 
  8. Uyuklu M, Canpolat M, Meiselman HJ, Baskurt OK. Wavelength selection in measuring red blood cell 
aggregation based on light transmittance. J Biomed Optics 20121;16:117006. 
  9. Lee BK, Durairaj A, Mehra A, Wenby RB, Meiselman HJ, Alexy T. Microcirculatory dysfunction in 
cardiac syndrome X: role of abnormal blood rheology. Microcirc 2008;15:451-460. 
10. Perelman N, Selvaraj SK, Batra S, Luck LR, Erdreich-Epstein A, Coates TD, Kalra VK, Malik P. 
Placenta growth factor activates monocytes and correlates with sickle cell disease severity. 
Blood 2003;102:1506-1514. 
11. Giri RK, Selvaraj SK, Kalra VK. Amyloid peptide-induced cytokine and chemokine expression in 
THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger 
RNA. J Immunol 2003;170:5281-5294. 
12. Giri R, Shen Y, Stins M, Yan SD, Schmidt AM, Stern D, Kim KS, Zlokovic B, Kalra VK. β-Amyloid-
induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1. Am J 
Physiol Cell Physiol 2000;279:C1772-C1781. 
13. Selvaraj SK, Giri RK, Perelman N, Johnson C, Malik P, Kalra VK. Mechanism of monocyte activation 
and expression of proinflammatory cytochemokines by placenta growth factor. Blood 2003;102:1515-1524. 
14. Giri RK, Rajagopal V, Shahi S, Zlokovic BV, Kalra VK. Mechanism of amyloid peptide induced 
CCR5 expression in monocytes and its inhibition by siRNA for Egr-1. Am J Physiol Cell Physiol 
2005;289:C264-C276. 
15. Lipid Research Clinics Program. The Manual of Laboratory Operations: Lipid and Lipoprotein 
Analysis. Bethesda, Md, National Institutes of Health, DHEW Publication No. NIH 75-628, 1974. 
16. Burstein M, Scholnick HR, Morfin R. Rapid method for the isolation of lipoproteins from human 
serum by precipitation with polyanions. J Lipid Res 1970;11:583-595. 
17. Freidewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein 
cholesterol in plasma without the use of the preparative ultracentrifuge. Clin Chem 1972;18:499-502. 
18. Nyholt DR, Yu C-E, Visscher PM. On Jim Watson’s APOE status: genetic information is hard to hide. 
Eur J Hum Genetics 2009;17:147-150. 
19. Callaway CW, Chumlea WC, Bouchard C. Circumferences. In: Lohman TG, Roche AF, Martorell R 
(eds). Anthropometric Standardization Reference manual. Champaign, IL: Human Kinetics Books, 
1988:39-47. 
20. Larsson B, Svardsudd K, Welin L, Wilhelmsen L, Bjorntorp P, Tibblin G. Abdominal adipose tissue 
distribution, obesity, and risk of cardiovascular disease and death: 13 year follow up of participants in the 
study of men born in 1913. Brit Med J 1984;288:1401-1401. 
21. Hartz A, Grubb B, Wild R. The association of waist hip ratio and angiographically determined 
coronary artery disease. Int J Obesity 1990;14:657-665. 

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8.0 
 
CAROTID ULTRASONOGRAPHIC PROTOCOL 
 
8.1  Summary of Carotid Artery Ultrasound Data Acquisition 
 
a) Far wall intima-media thickness (IMT) of the right distal common carotid artery (CCA) 
b) Diameter of the right distal common carotid artery 
 
8.2  Overview of Ultrasound Image Acquisition 
 
B-mode carotid artery images for IMT measurement are acquired with a Mindray DC 7 Digital 
Diagnostics Ultrasound System using a linear array L7-3 (frequency range: 3-7 MHz) transducer. The 
preset feature is used to enable image capture with optimized configuration of imaging parameter 
settings for this transducer and specific examination. Real-time dynamic clips are simultaneously 
captured along with a single-lead electrocardiogram (ECG) tracing and are stored as digital dynamic 
clips so that the ECG tracings can be viewed along with the B-mode images. The Mindray DC 7 can 
resolve 2 echo interfaces that are separated by 300 μm or more (in MARS (1), EPAT (2), VEAPS (3), 
BVAIT (4), WISH (5) and the general population (6) adult IMT is >400 μm). 
 
Participant ID, visit number, scan date and study name (NAPS) are recorded at the beginning of the 
procedure. Five sequential blood pressure measurements are recorded at the end of the procedure using a 
GE-CARESCAPE V100 vital signs monitor. The average systolic and diastolic blood pressure 
measurements are required along with the carotid arterial diameters during systole and diastole to 
calculate central vascular stiffness (Section 8.6). All ultrasound examinations are performed by an 
experienced ultrasonographer. The following standardized image acquisition approach is conducted with 
each ultrasound examination. 
 
Carotid artery intima-media thickness and carotid artery diameter imaging 
 
Participants are placed supine and positioned in a 45o molded head block with the neck extended to 
present the optimal angle for ultrasound examination of the right carotid artery. Using B-mode, the right 
CCA is imaged in cross section and the transducer moved laterally until the jugular vein and CCA are 
stacked with the former above the latter. In this position, the central image line passes along the 
common diameter of both vessels. 
 
 
 
Stacked vessels in cross section view 
 
Jugular Vein 
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The transducer is slowly rotated 90o around the central image line of the stacked jugular vein and CCA, 
maintaining the jugular vein stacked above the CCA while obtaining a longitudinal image of both 
vessels. This will ensure that the transducer remains perpendicular to the far wall in the longitudinal 
view. In this view, the jugular vein and carotid artery are in horizontal orientation. Maintaining light 
transducer pressure, an image including the proximal portion of the carotid bulb as well as the mid- to 
distal-portion of the CCA is obtained. The lowest overall gain is adjusted to obtain the optimal image. 
Both the near and far wall CCA IMT should be clearly visible; slight transducer angle adjustment may 
be necessary. Emphasis is placed on optimizing visualization of the IMT. Emphasis is then placed on 
optimizing visualization of the distal CCA luminal diameter in systole and diastole by simultaneously 
capturing both the CCA near and far wall IMT in the ultrasound image.  
 
 
 
Stacked vessels in longitudinal view 
 
The proximal portion of the carotid bulb is included in all images as an anatomical reference point for 
standardization of CIMT measurements. Stacking the jugular vein and CCA determines a repeatable 
transducer angle that allows the same portion of the wall to be imaged at each ultrasound examination 
(7,8). This leads to further standardization of image acquisition and processing that in turn decreases 
measurement variability (7,8). Minimum gain necessary for clear visualization of structures is used. 
Images are acquired from the carotid bulb and internal carotid artery, but emphasis of ultrasound 
imaging is on the distal centimeter of the CCA because least variability occurs in this area (9). The CCA 
far wall IMT is used for statistical purposes since measurement of near wall thickness is less accurate 
(10). The distal CCA is visualized longitudinally and 4-5 video clips (preset at 6 seconds in duration for 
each clip) are captured using the optimal transducer angle (OTA) view. While maintaining the optimized 
longitudinal image of the CCA, the transducer is moved away from the OTA view in the anterior 
direction and 1-2 video clips of CCA IMT images in the anterior view are captured. The transducer is 
then moved away from the OTA view in the posterior direction and 1-2 video clips of CCA IMT images 
in the posterior view are captured. 
 
The transducer is then moved cephalad (toward the head) maintaining the jugular vein stacked above the 
carotid artery to scan the carotid bulb (bifurcation) along the longitudinal plane. The transducer is swept 
to the anterior direction and then swept to the posterior direction bringing the far wall IMT of the 
bifurcation into view while maintaining the jugular vein stacked above the bifurcation. Emphasis is 
placed on optimizing visualization of the IMT and acquiring the thickest IMT image in the bifurcation 
view. A bifurcation view is defined as an image that contains 100% of the carotid bulb as well as 
visualization of the proximal internal or external carotid arteries; 1-2 video clips of the bifurcation view 
are captured. 
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Carotid artery bifurcation view 
 
Carotid artery plaque imaging 
 
Following acquisition of CIMT, the carotid artery is surveyed for plaques, defined as a focal thickening 
of the intima >1.5 mm. Beginning with the cross section view of the distal CCA, the transducer is 
moved cephalad from the distal CCA through the carotid bifurcation to the proximal internal and 
external carotid arteries then back to the distal CCA while surveying for lesions. The transducer is then 
slowly turned 90o to acquire a longitudinal plane view of the carotid artery. The transducer is then swept 
in the anterior direction and then swept in the posterior direction to acquire clear images of plaque(s), if 
any. If only one plaque is identified, the maximal carotid plaque thickness (MCPT) is obtained and 2 
video clips are captured. If multiple plaques are identified, each plaque is labeled (p1, p2, p3, etc.), the 
MCPT of each plaque is obtained and 1 video clip of each plaque is captured. 
 
 
 
Carotid artery plaque in the bifurcation view 
 
Longitudinal carotid artery imaging 
 
Repeat (follow-up) CIMT image acquisition uses the same ultrasound equipment and transducer during 
the entire project. Each individual’s baseline image of CIMT, arterial diameters and MCPT, if any are 
used as a guide to match the vascular and surrounding soft tissue structures of the follow-up ultrasound 
examinations. This is a direct visual aid method for reproducing transducer angulation designed for 
repeat image acquisition for longitudinal studies (1-5,7,8). In addition, repeat image acquisition 
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parameters must be the same as those used for baseline image acquisition, including transducer 
frequency range (MHz), depth, dynamic range, time gain controls (TGC), gain, power, etc., all of which 
are automatically recorded on the baseline ultrasound image and from which the setup parameters for 
the ultrasound equipment are derived. This procedure establishes standardization for instrument setup 
that encompasses the full dynamic range of the ultrasound echo across all examinations within the same 
participant. The key to repeat image reproducibility is to match all of the technical parameters and the 
follow-up image to the baseline image as precisely as possible. 
 
    
 
 
           CCA baseline image                                     CCA image 36 month later 
 
The primary trial end point is change in right distal CCA far wall IMT measured (over one centimeter) 
during end diastole. The transducer angle with the best definition of the blood-intima and 
media-adventitia echoes at baseline examination will therefore be used in all subsequent studies for 
quantitative measurement and comparison. The co-primary trial end point is change in carotid artery 
stiffness determined over serial systolic and diastolic distal CCA diameter measurements. 
 
Phantom images obtained from RMI 404 GS LE, Small Parts Gray Scale-Attenuation coefficients of 0.7 
db/cm/MHz are evaluated every 6 months to assure image quality and to standardize calibration pixel 
size for IMT and diameter measurements. In addition, if ultrasound equipment requires replacement or 
repair (beyond maintenance), phantom images are evaluated before performing ultrasound examinations. 
 
8.3  Carotid Ultrasound Image Acquisition and Standardization 
 
8.3.1  Equipment and Supplies 
 
1) 
Mindray DC 7 Digital Diagnostics Ultrasound System 
 
2) 
Linear array L7-3 (frequency range: 3-7 MHz) transducer 
 
3) 
Single-lead ECG – contained within the Mindray DC 7 Digital Diagnostics Ultrasound System 
 
4) 
ECG monitoring electrodes 
 
5) 
GE – V100 Vital Signs Monitor (automated blood pressure device) 
 
6) 
Phantom Test Instrument (RMI 404 GS LE, Small Parts Gray Scale-Attenuation coefficients of 0.7 

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db/cm/MHz) 
 
7) 
Examination table 
 
8) 
Ultrasound conducting gel 
 
9) 
45o molded head block 
 
10) Personal computer 
 
11) Back up USB flash drives and portable hard drives 
 
8.3.2  General Ultrasound Procedure and Variability 
 
The above overview (Section 8.2) and following details of our ultrasound techniques/methodology 
provide a high degree of standardization for image acquisition and processing, resulting in significant 
reduction in measurement variability between scans; inter- and intra-sonographer coefficients of 
variation (CVs) <3% (1-5,7,8). Even in multicenter studies with multiple acquisition sites, the 
methodology is highly stable and reproducible with low variability, <3% CV (11-13). 
 
8.3.3  Baseline CIMT Acquisition Procedure 
 
Carotid Artery (right) 
 
1) 
Turn on the Mindray DC 7 Digital Diagnostics Ultrasound System 
 
2) 
Select the study preset - NAPS 
 
3) 
Check the screen monitor to make sure that the preset parameter settings are as follows: 
 
- Transducer frequency - 7.0 MHz 
- Power input - 100 % 
- Dynamic range at mid-range - 55 
- Line density - M 
- FOV - W 
- Focus Number - 1 
- THI - off 
- iBeam - off 
- Smooth - 2 
- iClear - 4 
- Persist - 2 
- Gray Map - 5 
- Depth - 38 mm 
- Zoom mode - normal 
- ECG - on 
- TGC - should be fixed in mid-range position 
-  
4) 
Press NEW PATIENT key on the control panel to begin the CIMT examination and enter the 
following information on the screen monitor: 
 

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- Participant identification number (PID) 
- Date 
- Visit #  
- Study name (NAPS) 
- RT (scan of right carotid artery) or LT (scan of left carotid artery) 
- DEG - 45o (head angle) 
- PLN (head plain) - (enter NT, MT or CT during ultrasound examination) 
- Optimal transducer angle - (OTA, degree - enter 0, 15, 30, 45, 55, -5, -10, etc., during 
 
ultrasound examination) 
- OP - operator’s name 
 
5) 
Prepare the participant for scanning 
 
- Bring the participant into the ultrasound examination room. 
- Explain CIMT procedure to the participant. 
- Properly position participant in the supine position on the examination table. Remove 
necklaces and other objects that may interfere with the procedure. Participant's head and 
neck are anatomically aligned; place support pillows under participant's head, neck or 
shoulders if necessary. 
- Head is wedged between square pillow block on right and 45o wedge pillow on left. Left 
 
ear touches wedge pillow. Blocks rest upon shoulders. 
- Connect ECG leads: white - right wrist; black - left wrist; green - right leg. 
- Check ECG tracing on the screen monitor; adjust the ECG gain so that tracing is at the 
 
bottom of the screen and fully visible. 
 
6) 
Apply small amount of conducting gel to the field of scanning (right neck). Begin scanning. 
 
7) 
Begin scanning in the transverse plane on the proximal right side of the neck. Stack the right 
jugular vein and CCA. In this position, the cross-sections of both vessels appear near the center of 
image (do not capture the transverse image). 
 
8) 
Maintaining the transverse stacked image of the jugular vein and CCA, move the transducer 
towards the head. Move through the carotid bifurcation then back to the bulb-distal CCA area. 
 
9) 
Slowly rotate the transducer 90o degrees around the central image line of the stacked jugular vein 
and CCA, maintaining the jugular vein stacked above the CCA to a longitudinal image. In this 
position, the carotid artery image will be in a horizontal orientation. Instruct participant to perform 
a Valsalva maneuver to fully demonstrate the jugular vein and CCA, if necessary (do not capture 
this image). Keep transducer pressure light and obtain an image that contains the proximal portion 
of the carotid bulb as well as the mid- to distal- portion of the CCA. Adjust the overall gain to the 
lowest setting possible to obtain the optimal image (not during Valsalva). Both the near and far 
wall CCA IMT should be clearly visible.  
 
10) After the 90o transducer rotation, slight transducer angle adjustment may be necessary to obtain the 
longitudinal optimal transducer angle (OTA) view of the right CCA. Adjust the TGC’s and overall 
gain to obtain the optimal gain. Gain must be as low as possible (optimal gain) and stable 
throughout CIMT image acquisition. IMT will be brightest when the transducer angle is 
perpendicular to the vessel wall. 
 

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11) Simultaneously optimize the CCA IMT of the near and far walls. Enter from key board the degree 
of neck extension using one of the following anatomical landmarks as a perpendicular plane to the 
examination table: 
 
 
  
 
PLN (head plane) - enter any of  NT, MT or CT 
 
 
  
 
 
a) "NT" = tip of nose to external acoustic meatus 
 
  
 
 
b) "MT" = tip of mouth to the external acoustic meatus 
 
  
 
 
c) "CT" = tip of chin to the external acoustic meatus 
 
 
 
   
 
 
       a   b    c 
 
 
 
 
 
 
 
 
 
 
  
 
OTA, degree - enter any of 0,10,15,20,25,30,35,40,45,-5,-10, etc. 
 
  
  
 
 
 
a) 0 = transducer’s longitudinal view plane is parallel to the plane of the 
examination table 
 
  
 
 
 
b) 10 = transducer’s longitudinal view plane is 10o above the horizontal of the 
plane of the examination table 
 
  
 
 
 
c) -5 = transducer’s longitudinal view plane is 5o below the horizontal of the 
plane of the examination table 
 
12) Repeat steps 7-10 five times to ensure that the OTA image is a high quality CIMT image and 
repeatable during the scan. Capture at least 5 video clips of the OTA view. 
   
13) Annotate “ANT” on screen monitor. While maintaining the optimized longitudinal image of the 
CCA, move the transducer away from the OTA in the anterior direction to acquire CCA IMT 
images and capture 1-2 video clips of the anterior view.  
 
14) Annotate “POST” on screen monitor. Move the transducer away from the OTA in the posterior 
direction to acquire CCA IMT images and capture 1-2 video clips of the posterior view. 
 
15) Annotate “BIF” on screen monitor. Scan the longitudinal plane view of the bifurcation (carotid 
artery bulb). Sweep to the anterior direction and then sweep back to the posterior direction. Bring 
the far wall IMT of the bifurcation into view. Emphasis is placed on optimizing visualization of the 
IMT and acquiring the thickest IMT image on the bifurcation view. A bifurcation view is defined 
as an image that contains 100% of the carotid bulb as well as the proximal internal or external 
carotid arteries. Capture 1-2 video clips of the bifurcation view. Identify the internal and external 
carotid arteries by Doppler, if necessary. Annotate images to identify structures. 
 
16) Survey the carotid artery for plaques. In the cross section view, move the transducer cephalad 
(towards the head) from the distal CCA through the bifurcation to the proximal internal and 

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external carotid arteries then return to the distal DCC. Slowly rotate the transducer 90o to acquire a 
longitudinal plane view of the carotid artery. Sweep in the anterior direction then sweep in the 
posterior direction to acquire plaque(s) images, if any. If only one plaque is identified, obtain the 
maximal carotid plaque thickness (MCPT) and capture 2 video clips. If multiple plaques are 
identified, label each plaque (as p1,p2,p3…) and obtain the MCPT of each plaque and capture 1 
video clip of each plaque. Determine quantitative Doppler at site of all lesions. 
 
17) Annotate “Left” on screen monitor. Scan the left side CCA IMT by turning the participant’s head 
to the right and following steps 6-16. 
 
18) Standard output file format: Both AVI and DICOM video clips. Provide at least 16 clips if possible 
for each scan. This includes: 
 
 
 
5 clips - Longitudinal OTA views of the RCCA 
 
 
1 clip 
- Longitudinal anterior view of the RCCA 
 
 
1 clip 
- Longitudinal posterior view of the RCCA 
 
 
2 clips - Longitudinal views of the RBIF (IMT) 
 
 
2 clips - Longitudinal views of the MCPT, if any 
 
 
3 clips - Longitudinal OTA views of the LCCA 
 
 
1 clip 
- Longitudinal anterior view of the LCCA 
 
 
1 clip 
- Longitudinal posterior view of the LCCA 
 
 
2 clips - Longitudinal views of the RBIF (IMT) 
 
 
2 clips - Longitudinal views of the MCPT, if any 
 
19) Prepare the GE-V100 automated blood pressure device. Measure the participant’s upper right arm 
circumference to select the appropriate blood pressure cuff size. Snugly place the blood pressure 
cuff on the participant's right upper arm. Note the mark on the inside of the blood pressure cuff 
that needs to be placed over the brachial artery. Blood pressure cuff should fit snugly but not so 
tightly as to prevent venous return between determinations. Press POWER ON and then press 
INFLATE to begin the automated blood pressure determinations every one minute. Record five 
sequential blood pressure and pulse rate measurements on the participant’s data sheet. 
 
20) Completely and accurately complete the CIMT work sheet. 
 
21) Place a back-up USB flash drive or portable hard-drive to the ultrasound system’s tray. Click the 
[iStation] key on the control panel or click the [iStation] button in the review screen and select the 
source of the scan database, Find the participant’s PID and scan date in the review screen and 
select the examination that has been performed. Click [Send To] or [Back-up Patient Record] in 
the dialogue box and click [OK] or the [Back-Up] button in the dialogue box to begin writing to 
the system-supported media (Back-up USB flash drive or portable hard-drive). After the writing 
process is completed, click the symbol in the lower right corner of the monitor screen to eject the 
back-up USB flash drive or portable hard-drive. 
 
8.3.4  Repeat (Follow-up) CIMT Acquisition Procedure 
        
Use the same ultrasound equipment and transducer during entire CIMT project. Use participant’s 
baseline CCA, vascular diameter images, carotid artery bifurcation as well as MCPT images, if any as 
the reference for ultrasound equipment setup parameters and guidance for repeat image acquisition. 
Repeat image acquisition parameters must be the same as those used for baseline image acquisition 
including transducer frequency range (MHz), depth, dynamic range, TGC, gain, power, etc., all of which 

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are automatically recorded on the baseline ultrasound images. The key to image reproducibility is to 
match the follow-up image to the baseline image as precisely as possible. 
 
Follow the same CIMT acquisition procedure as used for baseline acquisition (See Section 8.3.3, 
Baseline CIMT Acquisition Procedure) and ensure that the follow up image landmarks look the same as 
those on the baseline image when performing follow-up image acquisition: 
 
- Top tissue pattern 
- Jugular vein pattern 
- Bottom tissue pattern 
- CCA near-wall IMT pattern 
- CCA far-wall IMT pattern 
- MCPT pattern, if any 
 
8.4  Automated Computerized Image Analysis of IMT 
 
A computer system developed to assess carotid arterial dimensions from ultrasound images includes a 
personal computer. Software developed to measure changes in arterial dimensions includes a technique 
for automated edge detection of the lumen-intima and media-adventitia echo lines for IMT 
measurements (1-5,7,8). Automated boundary detection provides IMT and luminal measurements with 
greater precision than manual boundary identification because multiple measurements along the vessel 
structures replace a single point measurement made with a cursor (7,8). When automated detected edges 
are observed they do not differ from those chosen by manual analysis although manual variability is 
considerably greater than that obtained from computer-detected gradient-based edges. A description of 
the critical parts of the image processing system is given below. 
 
Analysis of each B-mode carotid ultrasound frame involves the steps outlined below. 
 
1) Select the frame to be analyzed from the digital frame loop 
2) Enter the frame identification information (participant ID, date, etc.) 
3) Manually point to the approximate location of the lumen-intima and media-adventitia echoes for 
IMT measurements (or the adventitia-media and media-adventitia echoes for arterial diameter 
measurements) with a mouse 
4) Automatically track the lumen-intima and media-adventitia echoes and compute IMT (or 
automatically track the adventitia-media and media-adventitia echoes and compute arterial diameter) 
 
These steps are further discussed below. 
 
Frame selection: Ultrasound image input to the current ultrasound image processing and analysis 
workstation system is directly through the digital frame loops generated by the digital ultrasound 
imager. Digital frame loops are played back through the ultrasound image processing and analysis 
workstation system and optimum frames are selected through direct visualization of individual digitized 
frames within the digitized frame loop. 
 
Participant and measurement database: The program database has been structured to allow subjective 
descriptions of image quality and lesion morphology to be stored along with measurements and other 
process descriptors. The information stored is shown below. Data relating to image quality reflects on 
the relative reliability of each measurement and is taken into account when the measurements are 
analyzed. Participant and image information: 

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  Reader (computer operator) 
 
  Participant identification code 
 
  Visit number 
 
  Examination date 
 
  Frame time code 
 
 
Side  
 
(left,right) 
 
 
Segment code 
(CC1, CC2, CC3, BUL, ICA, PA, BA) 
 
 
Measurement code 
(near wall, far wall, lumen diameter) 
 
 
Tracking method 
(automatic, manual) 
 
 
Image quality 
 
 
Lumen-intima lesion? 
(yes,no) 
 
 
Wall characteristics, border 
 
 
Wall characteristics, density 
 
 
Wall characteristics, structure 
 
 
Double line visible? 
(yes,no) 
 
 
Measurement made? 
(yes,no) 
 
Ultrasound equipment parameter preset values for the ultrasound examination, such as transducer 
frequency range (MHz), depth, dynamic range, TGC, gain, power, etc. are automatically recorded on the 
ultrasound video clips, and are recorded separately to establish identical technical conditions for 
follow-up examinations. This process standardizes instrument setup that encompasses the full dynamic 
range of the ultrasound echo across all studies within the same participant. 
 
Automated boundary detection: Automatic detection of the lumen-intima and media-adventitia echo 
lines is difficult because the intensity of the boundary is highly variable and frequently discontinuous.  
Our software surmounts these difficulties in a three step edge detection process. Additionally, our 
computer tracking software searches for edges normal to the curvature of the artery.  
 
The first step is to identify the approximate location of the boundary at four to six points using a mouse. 
A smooth curve fit to these points serves as a guide to the automated edge tracking algorithm. 
 
In the second step of the edge tracking process, the computer examines image intensity values in the 
image along a series of paths perpendicular to the smooth curve. For each path, the point in the image 
where the intensity gradient is largest is conditionally labeled as the boundary of the echo along that 
path. For each boundary point, both the image coordinates and the gradient value are stored. 
 
The third step in the process compares the gradient value for each conditional edge to the maximum 
gradient value of all conditional edges of the boundary and eliminates those edges with gradient values 
less than a preselected fraction of the maximum value. This procedure eliminates weak edges and gaps 
from the boundary. 
IMT and lumen diameter measurement: The boundary tracking described above is applied to the 
lumen-intima and media-adventitia echoes. Since the longitudinal carotid images are nearly horizontal in 
the ultrasound pictures, wall thickness is measured along vertical image lines at each point of the wall 
where both boundary points exist. From these data, maximum and average wall thickness is computed.  
Lumen diameter is measured in the same way as the wall thickness with the boundary tracking applied 
to the near and far wall media-adventitia echoes. 
 
Lesion size measurements: The lumen-intima ultrasound echo from raised carotid artery lesions are 
frequently highly irregular and difficult to track with automated edge finding algorithms. In addition, 
calcium shadowing can obscure the media-adventitia boundary. To deal with these problems, an option 

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is available to use manual tracing of lesion boundaries with the mouse. When calcium shadowing 
completely obscures edges, the opposite common carotid artery is used. If this is also unreadable 
because of shadowing, and manual tracking is also impossible, the case is discarded. In our experience 
over tens of thousands of ultrasound examinations, this occurs in <%1 of participants. The type of 
tracking used (automatic or manual) is indicated in the database when such measurements are stored. 
 
From the resulting analyses by computer of the B-mode images, thickness of the far wall intima-media 
and arterial diameters are stored electronically. Our software automatically creates and stores averages, 
maxima and minima vessel dimensions. 
 
8.4.1  IMT Measurement Standardization 
 
Good IMT image processing derives from good image acquisition. 
 
All settings must be consistently and accurately shown along with the ultrasound scan images for each 
participant. This information is entered as part of the database during image processing. 
 
Equipment information: 
 
 
 
a) Transducer frequency range (MHz) 
 
 
b) Depth (field of view (FOV)) 
 
 
c) Dynamic range 
 
 
d) TGC 
 
 
e) Total gain 
 
 
f) Power 
 
 
g) Ultrasound instrument 
 
Participant information: 
 
 
 
a) Date of scan 
 
 
b) Visit number 
 
 
c) Participant ID 
 
 
d) Study (NAPS) 
 
 
e) Date of image processing 
 
 
f) Participant's positioning during ultrasound image acquisition 
 
 
g) Participant's side scanned (left, right) 
 
 
Image processing information: 
 
 
a) Segment analyzed 
 
 
CC1 
-  First cm from the proximal carotid artery bulb 
 
 
CC2  -  Second cm from the proximal carotid artery bulb 
 
 
CC3  -  Third cm from the proximal carotid artery bulb 
 
 
BUL  -  Carotid artery bulb 
 
 
ICA  -  Internal carotid artery 
 
 
b) Image processor's initials 
 
 
c) Frame number analyzed 
 
 
d) Ultrasound image description 
 
 
 
 
 
i) 
Angle variation of the common carotid artery 

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ii)    Distance of IMT measurement from the proximal carotid artery bulb 
 
 
 
 
 
iii)   Image name for storage 
 
 
e) Measurement code (near wall, far wall, lumen diameter) 
 
 
f) Tracking method (automatic, manual) 
 
 
g) Image quality 
 
 
h) Lesion notation 
Image processing calibration: 
 
a) This may vary from participant to participant (Depth or FOV) and by ultrasound imager 
 
b) Use the phantom test images to calibrate the horizontal and vertical pixel sizes 
 
Frame selection for image processing: 
 
a) Frames selected at QRS wave (R-S interval) and T-wave on each heart cycle (paired 
image) 
 
b) Frame selected where IMT is brightest and most continuous 
 
c) Must use the same referencing landmarks on each follow-up visit 
 
d) Name and save (at least 15 paired images) images 
IMT measurement: 
 
a) A pre-calibrated computer-generated 1 cm electronic ruler is placed above area of interest 
 
b) Using a mouse, points are marked directly above the lumen-intima echo along the 1 cm 
ruler length. This represents the first edge 
 
c) Using a mouse, points are marked directly below the media-adventitia echo along the 1 cm 
ruler length. This represents the second edge 
 
d) Steps a through c are repeated for the next consecutive frame (usually the next heart cycle) 
 
e) Based on the baseline measurement, the same measurement location is made on each 
follow-up scan (absolutely required) 
 
f) The image processed baseline scan is viewed during image processing of all follow-up 
scans to ensure step e 
8.5  Automated Computerized Arterial Diameter Analyses 
Procedures outlined in Section 8.4 are followed and applied to measurement of arterial diameter during 
systole (maximum arterial excursion) and diastole (minimum arterial excursion). Arterial diameter is 
defined from adventitia-media (near wall) to media-adventitia (far wall). Arterial diameter is measured 
along the same 1 cm segment identified by the computer-generated electronic ruler used for the 
measurement of IMT. 
8.6  Vascular Stiffness 
 
Measurements of right CCA lumen diameters at systole (DS) and diastole (Dd) are determined at the 
same site where CIMT is measured (8,14-16). Standardized brachial artery blood pressure measurements 
are determined using an automated oscillometric method (GE – V100 Vital Signs Monitor) at the end of 
each ultrasound examination (average of 5 supine measurements) in order to measure systolic (Ps) and 
diastolic (Pd) pressures and pulse pressure (PP=Ps-Pd) (8,14-16). The CV for Ds=1.28-2.33%, Dd=1.18-
2.23%, Ps=6.17-8.75% and Pd=6.96-8.77% (8,15). Two indices of arterial stiffness and arterial 
compliance are calculated as follows: 
 
 
 
Arterial distensibility = [2(Ds-Dd)/Dd]/PP 
 
 
Arterial compliance = (D2s-D2d)/PP 

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8.7  Quality Control 
 
These steps provide a measure of consistency of image analysis. 
 
1) The Mindray DC 7 Digital Diagnostics Ultrasound System - To monitor operation of Mindray 
DC 7 Digital Diagnostics Ultrasound System, a precision small parts grey scale phantom (RMI 
404 GS LE, Small Parts Gray Scale-Attenuation coefficients of 0.7 db/cm/MHz) simulating the 
lumen-intima and media-adventitia boundaries is scanned every 6 months. The distance between 
vertical and horizontal targets is measured with our IMT assessment software. Since the true size 
of the phantom distances do not change and the power and gain of the scanner is kept constant, 
any change in the measured distances indicates a performance change in the ultrasound system 
that will be corrected. Results of these measurements are plotted and monitored for drift. 
 
2) Phantom images obtained from the RMI 404 GS LE, Small Parts Gray Scale-Attenuation 
coefficients of 0.7 db/cm/MHz scanning test are evaluated before the project initiates. 
Subsequently, phantom scanning tests are required every 6 months to assure image quality, 
image resolution and measurement accuracy (see above) as well as to standardize calibration 
pixel size for IMT and diameter measurements. In addition, if ultrasound equipment requires 
replacement or repair (beyond usual maintenance), a phantom scanning test is required. 
       
3) Phantom scan procedures: 
 
A) 
Fill the water dam on the top of the phantom equipment. Ensure that water temperature is between  
 
70-76oF. 
 
 
 
B) 
Set ultrasound equipment image depth to 38 mm. Use the same transducer that is used for the 
CIMT project. Set the probe’s frequency range to the same range as is set for the CIMT project. 
For example, if in the CIMT project the linear probe is set to 7.0 MHz, then in the phantom 
scanning test the transducer is set to 7.0 MHz. Likewise, if in the CIMT project the linear 
transducer is set to 7.5 MHz, then in the phantom scanning test the transducer is set to 7.5 MHz. 
 

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C) 
Acquire an image that has 4 horizontal pins and 4 curved-down pins. Set gain as low as possible. 
Obtain the best axial and lateral resolution for the target group. Capture a digital video clip. 
 
 
 
D) 
Acquire an image that has 4 horizontal pins and 6 vertical pins. Adjust transducer pressure to 
obtain all pins in the normal positions of the horizontal and vertical lines.  Move the focal zone 
slowly down the imaging field. Set gain as low as possible. The image background must be as dark 
as possible to display sharper horizontal and vertical pins. Bright background and blurred pins are 
not acceptable for the calibration procedure. Capture the best digital video image clip two times. 
 
 

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E) 
Acquire a transverse view of the vessel simulation image by adjusting the imaging gain. Turn the 
transducer 90o to get a vessel simulation image with optimal gain in the longitudinal view. Capture 
a digital video clip. 
 
 
 
 
F) 
Freeze image and measure vessel simulation diameter that is approximately 3.5 mm long. Capture 
the best digital image. 
 
 
 
G) 
Unfreeze the image to obtain the original vessel simulation image with optimal gain. Then change 
time gain to maximum. Capture the best digital video image. Change time gain to minimum. 
Capture the best digital video image. 
 
Longitudinal vessel 
simulation image 
Transverse vessel 
simulation image 

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H) 
Complete the phantom work sheet. 
 
I) 
Provide 6 video clips for each phantom test: 
 
 
1 video clip - Image that has 4 horizontal pins and 4 curved-down pins 
 
2 video clips - Image that has 4 horizontal pins and 6 vertical pins 
 
1 video clip - Longitudinal vessel simulation image with optimal gain 
 
1 video clip - Longitudinal vessel simulation image with maximum gain 
 
1 video clip - Longitudinal vessel image with minimum gain 
 
8.8  Data Security 
 
To assure security of data, processed images are stored electronically and maintained in locked facilities. 
 
8.9  Data Transfer 
 
Participant records are exported to Excel-readable files. Data are down-loaded into an Excel file and 
delivered to the DCC.
Maximum Gain  
Minimum Gain   

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8.10  References 
 
1. 
Hodis HN, Mack WJ, LaBree L, Selzer RH, Liu CR, Liu CH, Alaupovic P, Kwong-Fu H, Azen 
SP. Reduction in carotid arterial wall thickness using lovastatin and dietary therapy: a randomized, 
controlled clinical trial. Ann Intern Med 1996;124:548-556. 
2. 
Hodis HN, Mack WJ, Lobo RA, Shoupe D, Sevanian A, Mahrer PR, Selzer RH, Liu CR, Liu CH, 
Azen SP, for the Estrogen in the Prevention of Atherosclerosis Trial Research Group. Estrogen in the 
prevention of atherosclerosis: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 
2001;135:939-953. 
3. 
Hodis HN, Mack WJ, LaBree L, Mahrer PR, Sevanian A, Liu CR, Liu CH, Hwang J, Selzer RH, 
Azen SP, for the VEAPS Research Group. Alpha tocopherol supplementation in healthy individuals 
reduces low-density lipoprotein oxidation but not atherosclerosis: the Vitamin E Atherosclerosis 
Prevention Study (VEAPS). Circulation 2002;106:1453-1459. 
4. 
Hodis HN, Mack WJ, Dustin L, Mahrer PR, Azen SP, Detrano R, Selhub J, Alaupovic P, Liu CR, 
Liu CH, Hwang J, Wilcox AG, Selzer RH, for the BVAIT Research Group. High-dose B-vitamin 
supplementation and progression of subclinical atherosclerosis: a randomized controlled trial. Stroke 
2009;40:730-736. 
5. 
Hodis HN, Mack WJ, Kono N, Azen SP, Shoupe D, Hwang-Levine J, Petitti D, Whitfield-
Maxwell L, Yan M, Franke AA, Selzer RH, for the Women’s Isoflavone Soy Health Research Group. 
Isoflavone soy protein supplementation and atherosclerosis progression in healthy postmenopausal 
women: a randomized controlled trial. Stroke 2011;42:3168-3175. 
6. 
Howard G, Sharrett AR, Heiss G, Evans GW, Chambless LE, Riley WA, Burke GL for the ARIC 
Investigators. Carotid artery intimal-medial thickness distribution in general populations as evaluated by 
B-mode ultrasound. Stroke 1993;24:1297-1304. 
7. 
Selzer RH, Hodis HN, Kwong-Fu Helenann, Mack WJ, Lee PL, Liu CR, Liu CH, Blankenhorn 
DH. Evaluation of computerized edge tracking for quantifying intima-media thickness of the common 
carotid artery from B-mode ultrasound images. Atherosclerosis 1994;111:1-11. 
8. 
Selzer RH, Mack WJ, Lee PL, Kwong-Fu H, Hodis HN. Improved common carotid elasticity and 
intima-media thickness measurement from computer analysis of sequential ultrasound frames. 
Atherosclerosis 2001;154:185-193. 
9. 
O'Leary DH, Bryant FA, Goodison MW, Rifkin MD, Gramiak R, Ball M, Bond MG, Dunn RA, 
Goldberg BB, Toole JF, Wheeler HG, Gustafson NF, Ekholm S, Raines JK. Measurement variability of 
carotid atherosclerosis: real-time (B-mode) ultrasonography and angiography. Stroke 
1987;18:1011-1017. 
10. Wendelhag I, Gustavsson T, Suurkula M, Berglund G, Wikstrand J. Ultrasound measurement of 
wall thickness in the carotid artery: fundamental principles and description of a computerized analysing 
system. Clin Physiol 1991;11:565-577. 
11. Currier JS, Kendall MA, Henry WK, Alston-Smith B, Torriani FJ, Tebas P, Li Y, Hodis HN, for 
the ACTG 5078 Study Team. Progression of carotid artery intima-media thickening in HIV-infected and 
uninfected adults. AIDS 2007;21:1137-1145. 
12. Currier J, Kendall MA, Zackin R, Henry K, Alston-Smith B, Torriani FJ, Schouten J, Mickelberg 
K, Li Y, Hodis HN, for the AACTG 5078 Study Team. Carotid artery intima-media thickness and HIV 
infection: traditional risk factors overshadow impact of protease inhibitor exposure. AIDS 2005;19:927-
933. 
13. Kaplan RC, Kingsley LA, Gange SJ, Benning L, Jacobson LP, Lazar J, Anastos K, Tien PC, 
Sharrett AR, Hodis HN. Low CD4+ T-cell count as a major atherosclerosis risk factor in HIV-infected 
women and men. AIDS 2008;22:1615-1624. 
14. Seaberg EC, Benning L, Sharrett AR, Lazar JM, Hodis HN, Mack WJ, Siedner MJ, Phair JP, 
Kingsley LA, Kaplan RC. Association between human immunodeficiency virus infection and stiffness 
of the common carotid artery. Stroke 2010;41:2163-2170. 

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15. Kaplan RC, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, Xue X, Parrinello CM, Hunt 
P,  Deeks SG, Hodis HN. T-cell activation and senescence predict carotid artery stiffness among HIV-
infected women. Atherosclerosis 2011;217:207–213. 
16. Mack WJ, Dhungana B, Dowsett SA, Keech CA, Hodis HN. Carotid artery intima-media thickness 
after raloxifene treatment. J Women’s Health 2007;16:370-378. 
 

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9.0 
DATA COLLECTION AND MANAGEMENT 
 
9.1  Study Personnel and Contact Information 
 
9.1.1  Investigators 
 
1) 
Howard N. Hodis, M.D.  (323) 442-1478, athero@usc.edu 
 
2) 
Vijay Kalra, Ph.D.  (323) 442-1526 or (626) 388-5924, vkalra@usc.edu 
 
3) 
Howard A. Liebman, M.D.  (323) 865-3950, liebman_h@med.usc.edu 
 
4) 
Wendy J. Mack, Ph.D.  (323) 442-1820, wmack@usc.edu 
 
5) 
Herbert J. Meiselman, Sc.D.  (323) 442-1268 or (323) 442-1267, hmeiselman@gmail.com or 
meiselman@usc.edu 
 
9.1.2  Data Coordinating (DCC) Personnel 
 
1) 
DCC Director:  Wendy Mack, Ph.D.  (323) 442-1820, wmack@usc.edu 
 
2) 
Data Programmer/Analyst:  Laurie Dustin, M.S.  (323) 442-1566, labree@usc.edu 
 
3) 
Data Programmer/Analyst:  Naoko Kono, M.S.  (323) 442-1802, kono@usc.edu 
 
4) 
Data Monitor:  Olga Morales  (323) 442-1568, molga@usc.edu 
 
5) 
Data Systems Engineer:  Adrian Herbert (323) 442-2248, aherbert@usc.edu 
 
9.1.3  Clinic Personnel 
 
1) 
Study Coordinator:  Liny Zurbrugg, R.N.  (323) 442-2425 
 
2) 
Patient Coordinator: TBA  (323) 442-1754 
 
3) 
Study Nurse:  TBA  (323) 442-2257 
 
4) 
Phlebotomist:  TBA  (323) 442-1488 
 
5) 
Phlebotomist:  Esther Bhimani  (323) 442-2014 
 
6) 
Project Coordinator:  Janie Teran  (323) 442-1478; FAX (323) 442-2685, athero@usc.edu 
 
7) 
Clinic FAX:  (323) 442-2345 
 
9.1.4  Ultrasound Personnel 
 
1) 
Yanjie Li, M.D.  (323) 442-3993, yanli@usc.edu 

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2) 
Mingzhu Yan, M.D., Ph.D.  (323) 442-1303, mingzhuy@usc.edu 
 
3) 
FAX:  (323) 442-2345 
 
9.1.5  Participating Laboratories 
 
1) 
Quest Corporation 
 
Contact Person:  Ellie Hetman  (818) 737-8347 
 
To measure chemistry panel, insulin, complete blood count (CBC) with platelet count, 
prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen (see Section 
7). 
 
2) 
USC Hematology Laboratory 
 
Laboratory Director:  Howard A. Liebman, M.D.  (323) 865-3950, liebman_h@med.usc.edu 
 
Contact Person:  Leanne Rochanda, (323) 865-0965, rochanda@usc.edu 
 
To measure vWF antigen, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 
(PAI-1), D-dimers, platelet aggregation (ADP, collagen, epinephrine), ristocetin cofactor, factor 
VIII, plasma nattokinase levels and anti-nattokinase antibodies (see Section 7). 
 
3) 
USC Rheology Laboratory 
 
 
Laboratory Director: Herbert J. Meiselman, Sc.D.  (323) 442-1268 or (323) 442-1267, 
hmeiselman@gmail.com or meiselman@usc.edu 
 
 
Contact Person:  Rose Wenby, (323) 442-1268 or (323) 442-1267, rwenby@usc.edu 
 
To measure erythrocyte sedimentation rate (ESR), red blood cell aggregation (plasma), red blood 
cell aggregability, plasma viscosity and whole blood viscosity (see Section 7). 
 
4) 
USC Biochemistry Laboratory 
 
 
Laboratory Director:  Vijay Kalra, Ph.D. (323) 442-1526, vkalra@usc.edu 
 
Contact Person:  Caryn Gonsalves, Ph.D. (323) 442-1528 or (626) 353-8232 
 
To measure MCP-1, IL-8, TNFα, IL-1β, IL-10, E-selectin, P-selectin, endothelial injury in-vitro 
assay: VCAM-1, ICAM-1 and monocyte activation (FACS): CD11b, CD11c, VLA-4 (see Section 
7). 
 
5) 
USC Atherosclerosis Research Unit (ARU) Core Lipid Laboratory 
 
Laboratory Director:  Juliana Hwang, Pharm.D.  (323) 442-1160 or (323) 442-2297, 
julianah@usc.edu 
 

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Contact Person:  Gail Izumi  (323) 442-1160 or (323) 442-2297, izumi@usc.edu 
 
To measure lipid, insulin and hemoglobin A1c levels as well as prepare samples for -80oC storage 
(see Section 7). 
 
9.2  Laboratory Data 
 
Because the study is double-blinded, all laboratory data except Quest safety data (chemistry, CBC with 
platelets, PT, aPTT and fibrinogen) will be entered by laboratory personnel directly into the trial data 
system; clinic personnel will be blinded to these laboratory data. Pre-randomization screening values for 
LDL-C will be made available to the study clinic personnel. On-trial LDL-C levels will also be routinely 
reported to the clinic. 
 
9.2.1  Sample Labeling and Tracking 
 
Bar-code labeling will be used for collection, processing, storage and disposition of all laboratory 
samples by the following procedure: 
 
1) 
The samples to be collected at each clinic visit will be listed in pre-made requisition forms, 
generated through the trial data system; the requisition form will contain pre-printed participant ID, visit 
number (linking visit number to samples to be collected), number and type of blood tubes to be used for 
that particular clinic visit and a bar code. 
 
2) 
Sample collection tube labels will be generated at the study clinic using the trial data system. On 
the initial collection tubes, labels will include participant ID, visit number and the type of tube and 
specimen collected, thus assuring that the proper sample is collected in the appropriate collection tube 
for each clinic visit. 
 
3) 
Receipt of samples by staff at the Core Lipid Laboratory will be compared and verified against the 
bar-coded laboratory requisition form that shows which samples should be received according to clinic 
visit. Any missing samples will be immediately brought to the attention of the clinic staff. 
 
4) 
The Core Lipid Laboratory staff will use the trial data system to generate bar-coded labels 
(cryolabels for stored samples) for each tube. The bar code will contain data on participant ID, visit 
number, type of sample collected and a laboratory ID number. 
 
5) 
In addition to the data included in the bar code, the sample data table in the trial database will 
include date of sample collection, the directed analyte(s) for future measurements of stored samples, 
storage location (for stored samples), as well as information on ultimate disposition and receipt of the 
sample (date sent, date received, to what laboratory, comments), thereby serving as the laboratory log as 
to the receipt and disposition of all samples. 
 
6) 
A sample flow-sheet that shows the number and type of samples to be stored at -80oC  will be used 
by Core Lipid Laboratory staff to compare and verify against the pre-made requisition forms for each 
clinic visit. 
 
7) 
Personnel from the following laboratories will enter their respective laboratory measurements 
directly into the sample data table: Core Lipid Laboratory, Hematology Laboratory, Rheology 
Laboratory, Biochemistry Laboratory. 
 

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8) 
The DCC will run routine checks of the laboratory data tables to determine whether and which 
data may be missing. If data are missing because of shipment loss or sample loss at the laboratory level, 
back-up storage samples will be re-shipped to the appropriate laboratory so that the analyte can be 
measured. 
 
9.2.2  Lipid, C-Reactive Protein (CRP) and Hemoglobin A1c Data 
 
Samples will be analyzed by the ARU Core Lipid Laboratory to obtain lipid, CRP and hemoglobin A1c 
data. The contact person at this laboratory is: 
 
Gail Izumi, (323) 442-1160 or (323) 442-2297, izumi@usc.edu 
 
9.2.2.1  Data Transmission 
 
All data will be entered directly into the trial data system by personnel at the Core Lipid Laboratory. A 
random sample of hard copies of data will be requested by the DCC for double entry and estimation and 
monitoring of data entry error rates. 
 
9.2.2.2  Study Data 
 
The following data will be obtained: 
 
A) Lipids (precipitation method): 
 
   
1) total plasma cholesterol 
   
2) total plasma triglycerides 
   
3) plasma HDL-C 
   
4) calculated VLDL-C 
   
5) calculated LDL-C 
 
LDL-C will be calculated by the Friedewald equation: 
 
LDL = Total Cholesterol - HDL-cholesterol - (Triglycerides/5) 
 
This equation is applicable only when triglycerides are less than 500 mg/dl. When triglycerides are 
greater than this value, the calculated LDL-C will be set to missing in the lipid database. Calculated 
LDL-C and VLDL-C (Triglycerides/5) will be computed by the DCC after data entry of laboratory 
lipids. 
 
B) Metabolic 
 
 
 
 
6) C-reactive protein 
 
 
 
7) hemoglobin A1c 
 
9.2.2.3  Data Collection Schedule 
 
1) 
Lipid data by the precipitation method will be assessed at the screening visit (SV) and v06 and 
every 6 months on-trial (study visits v12, v18, v24, v30, v36). 
 
2) 
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v12, v18, v24, v30, v36). 
 
3) 
Hemoglobin A1c will be obtained at SV, v03 and v06 and every 6 months on-trial (study visits 
v12, v18, v24, v30 and v36). 
 
9.2.2.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send samples to the Core Lipid Laboratory. 
 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition forms and tube labels. 
 
3) 
Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 
9.2.2.5  ARU Core Lipid Laboratory Responsibilities 
 
1) 
Receipt and storage of samples from phlebotomist. 
 
2) 
Compare and verify samples received against the bar-coded laboratory requisition form that shows 
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
missing samples. 
 
3) 
Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
 
4) 
Timely and accurate processing of laboratory data. 
 
5) 
Prepare data sheets with laboratory results and enter laboratory data of lipids, CRP and 
hemoglobin A1c into the trial database. 
 
6) 
Perform ongoing laboratory quality control and standardization procedures. 
 
7) 
Provide DCC with random samples of hard copies of laboratory data on DCC request. 
 
9.2.2.6  DCC Responsibilities 
 
1)  Review newly entered laboratory data for completeness and apparent accuracy. 
 
2)  Perform double data entry on random 10% of laboratory data. Compare double-entered files 
(entered at laboratory versus DCC). Calculate and report error rates in weekly data reports. Report 
unacceptably high error rates to Steering Committee. 
 
3)  Monitor timeliness of entry of data into trial data system. 
 
4)  Routinely inform laboratory of data not yet entered. 
 
5)  Compare participant's data with past data for possible within-subject outliers; generate queries to 
laboratory and monitor responses. 
 
6)  Monitor possible drift in laboratory values over time. 
 
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8)  Edit database as appropriate in response to data queries. 
 
9.2.3  Hematology Laboratory Data 
 
Samples will be analyzed by the Hematology Laboratory to obtain coagulation factors (vWF antigen, 
tPA, PAI-1, D-dimers), platelet aggregation (ADP, collagen, epinephrine), ristocetin cofactor, factor 
VIII, plasma nattokinase levels and anti-nattokinase antibodies. The contact person at this laboratory is: 
 
Leanne Rochanda, (323) 865-0965, rochanda@usc.edu 
 
9.2.3.1  Data Transmission 
 
All data will be entered directly into the trial data system by personnel at the Hematology Laboratory. A 
random sample of hard copies of data will be requested by the DCC for double entry and estimation and 
monitoring of data entry error rates. 
 
9.2.3.2  Study Data 
 
The following data are obtained: 
 
A) Coagulation factors: 
 
1) vWF antigen 
2) tPA 
3) PAI-1 
4) D-dimers 
5) factor VIII 
 
B) Platelet aggregation: 
 
 
   
6) ADP 
 
   
7) collagen 
 
   
8) epinephrine 
9) ristocetin cofactor 
 
C) Plasma product levels: 
 
 
   
10) nattokinase levels 
 
   
11) anti-nattokinase antibody 
 
9.2.3.3  Data Collection Schedule 
 
1) 
Coagulation factors and ristocetin cofactor will be assessed at baseline (v00), week 1, v01, v06 and 
every 12 months on-trial (study visits v18 and v30). 
 
2) 
Platelet aggregation will be assessed at baseline (v00), v01 and v06. 
 
3) 
Nattokinase levels will be determined at baseline (v00) and at every subsequent on-trial clinic 
visit; every month for the first 6 months and then every 3 months for the remainder of the trial 
(study visits week 1, v01, v02, v03, v04, v05, v06, v09, v12, v15, v18, v21, v24, v27, v30, v33, 

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36). 
 
4) 
Anti-nattokinase antibodies will be determined at baseline (v00), v01, v06 and every 12 months on 
trial (study visits v18 and v30). 
 
9.2.3.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send samples to Core Lipid Laboratory. 
 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition form and tube labels. 
 
3) 
Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 
9.2.3.5  ARU Core Lipid Laboratory Responsibilities 
 
1) 
Receipt of samples from phlebotomist.   
 
2)      Compare and verify samples received against the bar-coded laboratory requisition form that shows  
 
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
 
missing samples. 
 
3)      Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
 
4) 
Deliver samples to Hematology Laboratory and enter delivery into trial data system. 
 
9.2.3.6  Hematology Laboratory Responsibilities 
 
1) 
Receive samples from Core Lipid Laboratory and enter receipt into trial data system. 
 
2) 
Timely and accurate processing of laboratory data. 
 
3) 
Prepare data sheets with laboratory results and enter into the trial database. 
 
4) 
Perform ongoing laboratory quality control and standardization procedures. 
 
5) 
Provide DCC with random samples of hard copies of laboratory data on DCC request. 
 
9.2.3.7  DCC Responsibilities 
 
1)  Review newly entered laboratory data for completeness and apparent accuracy. 
 
2)  Perform double data entry on random 10% of laboratory data. Compare double-entered files 
(entered at laboratory versus DCC). Calculate and report error rates in weekly data reports. Report 
unacceptably high error rates to Steering Committee. 
 
3)  Monitor timeliness of entry of data into trial data system. 
 
4)  Routinely inform laboratory of data not yet entered. 
 
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laboratory and monitor responses. 
 
6)  Monitor possible drift in laboratory values over time. 
 
7)  Inform laboratory director and laboratory personnel of outliers and data drift in weekly reports. 
 
8)  Edit database as appropriate in response to data queries. 
 
9.2.4  Rheology Laboratory Data 
 
Samples will be analyzed by the Rheology Laboratory to obtain rheology measures (ESR, red blood cell 
aggregation (plasma), red blood cell aggregability (polymer), plasma viscosity, whole blood viscosity). 
The contact person at this laboratory is: 
 
Rose Wenby, (323) 442-1268 or (323) 442-1267, rwenby@usc.edu 
 
9.2.4.1  Data Transmission 
 
All data will be entered directly into the trial data system by personnel at the Rheology Laboratory. A 
random sample of hard copies of data will be requested by the DCC for double entry and estimation and 
monitoring of data entry error rates. 
 
9.2.4.2  Study Data 
 
The following data will be obtained: 
 
A) 
Rheology measures: 
 
 
 
1) ESR 
 
 
2) red blood cell aggregation (plasma) 
 
 
3) red blood cell aggregability (polymer) 
 
 
4) plasma viscosity 
 
 
5) whole blood viscosity 
 
9.2.4.3  Data Collection Schedule 
 
1) 
Rheology measures are assessed at baseline (v00), week 1, v01, v06 and every 12 months on-trial 
(study visits v18 and v30). 
 
9.2.4.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send samples to Core Lipid Laboratory. 
 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition form and tube labels. 
 
3) 
Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 
9.2.4.5  ARU Core Laboratory Responsibilities 
 
1) 
Receipt of samples from phlebotomist. 

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2) 
Compare and verify samples received against the bar-coded laboratory requisition form that shows  
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
missing samples. 
 
3) 
Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
 
4) 
Deliver samples to Rheology Laboratory and enter delivery into trial data system. 
 
9.2.4.6  Rheology Laboratory Responsibilities 
 
1) 
Receive samples from Core Lipid Laboratory and enter receipt into trial data system. 
 
2) 
Timely and accurate processing of laboratory data. 
 
3) 
Prepare data sheets with laboratory results and enter into the trial database. 
 
4) 
Perform ongoing laboratory quality control and standardization procedures. 
 
5) 
Provide DCC with random samples of hard copies of laboratory data on DCC request. 
 
9.2.4.7  DCC Responsibilities 
 
1) 
Review newly entered laboratory data for completeness and apparent accuracy. 
 
2) 
Perform double data entry on random 10% of laboratory data. Compare double-entered files 
(entered at laboratory versus DCC). Calculate and report error rates in weekly data reports. Report 
unacceptably high error rates to Steering Committee. 
 
3) 
Monitor timeliness of entry of data into trial data system. 
 
4) 
Routinely inform laboratory of data not yet entered. 
 
5) 
Compare participant's data with past data for possible within-subject outliers; generate queries to 
laboratory and monitor responses. 
 
6) 
Monitor possible drift in laboratory values over time. 
 
7) 
Inform laboratory director and laboratory personnel of outliers and data drift in weekly reports. 
 
8) 
Edit database as appropriate in response to data queries. 
 
9.2.5  Biochemistry Laboratory Data 
 
Samples will be analyzed by the Biochemistry Laboratory to obtain plasma inflammatory markers 
(MCP-1, IL-8, TNFα, IL-1β, IL-10, E-selectin, P-selectin), endothelial injury in-vitro assay 
inflammatory markers (VCAM-1, ICAM-1) and monocyte activation (FACS) markers (CD11b, CD11c, 
VLA-4). The contact person at this laboratory is: 
 
Caryn Gonsalves, Ph.D. (323) 442-1528 or (626) 353-8232 
 

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9.2.5.1  Data Transmission 
 
All data will be entered directly into the trial data system by personnel at the Biochemistry Laboratory.  
A random sample of hard copies of data will be requested by the DCC for double entry and estimation 
and monitoring of data entry error rates. 
 
9.2.5.2  Study Data 
 
The following data will be obtained: 
 
A) 
Plasma inflammatory markers: 
 
1) MCP-1 
2) IL-8 
3) TNFα 
4) IL-1β 
5) IL-10 
6) E-selectin 
7) P-selectin 
 
B) 
Endothelial injury in-vitro assay inflammatory markers: 
 
 
 
8) VCAM-1 
 
 
9) ICAM-1 
 
C) 
Monocyte activation (FACS) markers: 
 
 
 
10) CD11b 
 
 
11) CD11c 
 
 
12) VLA-4 
 
9.2.5.3  Data Collection Schedule 
 
1) 
Plasma inflammatory markers are assessed at screening visit (SV), v02, v09 and every 12 months 
on-trial (study visits v21 and v33). 
 
2) 
Endothelial injury in-vitro assay inflammatory markers are assessed at baseline (v00), v02, v09 
and every 12 months on-trial (study visits v21 and v33). 
 
3) 
Monocyte activation (FACS) markers are assessed at baseline (v00), v02, v09 and every 12 
months on-trial (study visits v21 and v33). 
 
9.2.5.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send samples to Core Lipid Laboratory. 
 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition forms and tube labels. 
 
3) 
Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 

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9.2.5.5  ARU Core Lipid Laboratory Responsibilities 
 
1) 
Receipt of samples from phlebotomist.   
 
2) 
Compare and verify samples received against the bar-coded laboratory requisition form that shows 
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
missing samples. 
 
3) 
Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
 
4) 
Deliver samples to Biochemistry Laboratory and enter delivery into trial data system. 
 
9.2.5.6  Biochemistry Laboratory Responsibilities 
 
1) 
Receive samples from Core Lipid Laboratory and enter receipt into trial data system. 
 
2) 
Timely and accurate processing of laboratory data. 
 
3) 
Prepare data sheets with laboratory results and enter into the trial database. 
 
4) 
Perform ongoing laboratory quality control and standardization procedures. 
 
5) 
Provide DCC with random samples of hard copies of laboratory data on DCC request. 
 
9.2.5.7  DCC Responsibilities 
 
1) 
Review newly entered laboratory data for completeness and apparent accuracy. 
 
2) 
Perform double data entry on random 10% of laboratory data. Compare double-entered files 
(entered at laboratory versus DCC). Calculate and report error rates in weekly data report. Report 
unacceptably high error rates to Steering Committee. 
 
3) 
Monitor timeliness of entry of data into trial data system. 
 
4) 
Routinely inform laboratory of data not yet entered. 
 
5) 
Compare participant's data with past data for possible within-subject outliers; generate queries to 
laboratory and monitor responses. 
 
6) 
Monitor possible drift in laboratory values over time. 
 
7) 
Inform laboratory director and laboratory personnel of outliers and data drift in weekly report. 
 
8) 
Edit database as appropriate in response to data queries. 
 
9.2.6  Quest Laboratory 
 
Chemistry panel, insulin, CBC, platelet count, PT, aPTT and fibrinogen data are analyzed by Quest. 
Because these laboratory results constitute clinical safety data are not crucial to blinding of study 
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photocopy of the laboratory results is made and sent to the DCC. 
 
The contact person in this laboratory is: 
 
Ellie Hetman  (818) 737-8347 
 
9.2.6.1  Data Transmission 
 
All data are sent from Quest to the study clinic. These data are reviewed by the research 
nurse/phlebotomist and abnormal values reported to Dr. Hodis. 
 
9.2.6.2  Study Data 
 
The following data will be obtained: 
 
A) Chemistry: 
 
  1) albumin 
 
 
12)    GGTP 
  2) albumin/globulin ratio 
13)    glucose 
  3) alkaline phosphatase 
14)    iron 
  4) ALT (SGPT) 
 
 
15)    LDH 
  5) AST (SGOT) 
 
 
16)    phosphorous 
  6) bicarbonate 
 
 
17)    potassium 
  7) BUN 
 
 
18)    sodium 
  8) BUN/creatinine ratio 
19)    total bilirubin 
  9) calcium 
 
 
20)    total protein 
10) chloride 
 
 
21)    uric acid 
11) creatinine 
 
 
 
B) Metabolic 
 
22)    insulin 
 
B) Complete blood count: 
 
23) WBC 
 
 
28)    MCH 
24) RBC 
 
 
29)    MCHC 
25) hemoglobin 
 
 
30)    RDW 
26) hematocrit 
 
 
31)    platelet count 
27) MCV 
 
C) Clotting times: 
 
 
 
32) prothrombin time 
 
 
33) activated partial thromboplastin time 
 
 
34) fibrinogen 
 
 
 
 
 

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9.2.6.3  Data Collection Schedule 
 
1) 
Chemistry panel is determined at screening visit (SV), v01 and every month for 6 months (study 
visits v02, v03, v04, v05, v06) and then every 6 months on-trial (study visits v12, v18, v24, v30 
and v36). 
 
2) 
Fasting plasma insulin is determined at screening visit (SV), v03, v06 and then every 6 months on-
trial (study visits v12, v18, v24, v30 and v36). 
 
3) 
CBC and platelet count data are assessed at screening visit (SV), at v01 and every month for 6 
months (study visits v02, v03, v04, v05, v06) and then every 6 months on-trial (study visits v12, 
v18, v24, v30 and v36). 
 
4) 
PT and aPTT are determined screening visit (SV), v01, v03 and v06 and every 6 months on-trial 
(study visits v12, v18, v24, v30, v36). 
 
5) 
Fibrinogen is determined at baseline (v00), week 1, v01 and v06 and every 6 months on trial (study 
visits v12, v18, v24, v30 and v36). 
 
9.2.6.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send samples to Core Lipid Laboratory. 
 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition forms and tube labels. 
 
3)  Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 
4)  Receive data from Quest laboratory; enter receipt date into trial data system. 
 
5)  Use DCC weekly reports to monitor timeliness of receipt of data. 
 
6)  Routinely inform Quest laboratory of data not yet received. 
 
7)  Send hard copy of data to the DCC data monitor. 
 
8)  Review data and inform Dr. Hodis of abnormal values. 
 
9)  Enter abnormal values onto Adverse Event log and complete Adverse Event form. 
 
9.2.6.5  ARU Core Lipid Laboratory Responsibilities 
 
1) 
Receipt of samples from phlebotomist. 
 
2) 
Compare and verify samples received against the bar-coded laboratory requisition form that shows 
 
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
 
missing samples. 
 
3) 
Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
 
4) 
Place samples in Quest pick up box for messenger to deliver to Quest Laboratory. 

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5) 
Enter delivery into trial data system. 
 
9.2.6.6  Quest Laboratory Responsibilities 
 
1) 
Timely and accurate processing of laboratory data. 
 
2) 
Send laboratory results to ARU Research Clinic. 
 
9.2.6.7  DCC Responsibilities 
 
1)  Receive data from clinic and review for completeness and apparent accuracy. 
 
2)  Perform double data entry on random 10% of laboratory data. Compare double-entered files 
(entered at laboratory versus DCC). Calculate and report error rates in weekly data report. Report 
unacceptably high error rates to Steering Committee. 
 
3)  Monitor timeliness of entry of data into trial data system. 
 
4)  Routinely inform clinic of data not yet entered. 
 
5)  Compare participant's data with past data for possible within-subject outliers; generate queries to 
laboratory and monitor responses. 
 
6)  Monitor possible drift in laboratory values over time. 
 
7)  Inform laboratory director and laboratory personnel of outliers and data drift in weekly reports. 
 
8)  Edit database as appropriate in response to data queries. 
 
9)  Monitor safety data and prepare routine reports on adverse laboratory values; confirm that 
abnormal laboratory values have been reported as Adverse Events. 
 
9.2.7  Sample Storage 
 
Sample storage will be conducted by the ARU Core Lipid Laboratory. The contact person at this 
laboratory is: 
 
Gail Izumi, (323) 442-1160 or (323) 442-2297, izumi@usc.edu 
 
9.2.7.1  Sample Tracking 
 
All sample storage information (i.e., that samples were collected and storage location) is tracked in the 
trial data system using bar coding of tubes. 
 
9.2.7.2  Study Data 
 
Serum and plasma (EDTA, citrate) samples are obtained for measurements to be determined at a future 
date: 
 

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1)  Fasting homocysteine and B-vitamins (vitamins B6 and B12 and folate) 
 
2)  Apolipoproteins 
a) Apo AI 
b) Apo B 
c) Apo E 
d) Apo CIII (total plasma) 
  
e) Apo CIII-HP 
  
f) Apo CIII-HS 
  
g) Apo CIII-HS/Apo CIII-HP ratio 
 
3)  Lipoprotein particles: 
a) LP-B 
b) LP-Bc 
c) LP-B:C + LP-B:C:E 
d) LP-AII:B:C:D:E (LP-AII:B complex) 
e) LP-AI 
f) LP-AI:AII 
 
4)  Lp(a) 
 
5)  Nitric oxide 
 
6)  Coagulation factors 
a) Activated protein C (APC) 
 
7)  Hormones 
a) Estradiol 
b) Estrone 
c) Estrone-sulfate 
d) Testosterone 
e) Progesterone 
f) Sex hormone binding globulin 
g) Prolactin 
 
8)  Bone markers 
a) Bone-specific alkaline phosphatase 
b) Osteocalcin 
c) Osteoprotegrin 
d) RANKL 
 
9)  Buffy coat 
 
    10)  Apolipoprotein E genotype 
 
9.2.7.3  Data Collection Schedule 
 
Blood samples for storage will be collected at baseline (v00) and at every subsequent clinic visit on-trial 
(study visits week 1, v01, v02, v03, v04, v05, v06, v09, v12, v15, v18, v21, v24, v27, v30, v33 and v36). 
 

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9.2.7.4  Clinic Responsibilities 
 
1) 
Phlebotomist to draw, label and send serum and plasma samples to Core Lipid Laboratory. 
2) 
Phlebotomist to use trial data system to generate pre-printed requisition forms and tube labels. 
 
3) 
Phlebotomist to indicate on trial data system date and samples sent to Core Lipid Laboratory. 
 
9.2.7.5  ARU Core Lipid Laboratory Responsibilities 
 
1) 
Receipt of samples from phlebotomist. 
 
2) 
Compare and verify samples received against the bar-coded laboratory requisition form that shows 
 
which samples should be received according to clinic visit. Immediately notify clinic staff of any 
 
missing samples. 
 
3) 
Use the trial data system to generate bar-coded labels (cryolabels for stored samples) for each tube. 
Storage samples contain labels with participant initials, participant identification number, date of 
sample collection, visit number, laboratory ID number, study name (NAPS) and the vial contents 
(EDTA Plasma, Citrated Plasma, Serum and Buffy Coat) and what future determination is to be 
performed with the sample. 
 
4) 
Timely aliquoting and properly storing plasma, serum and buffy coat samples. 
 
5) 
Store samples at -80oC in Core Lipid Laboratory. 
 
6) 
Enter all samples received for storage into trial data system. 
 
7) 
Create and maintain storage maps for all stored samples. 
 
9.3  Primary Study End Point Data:  Carotid Ultrasound Data 
 
The following ultrasound data will be obtained: 
 
1)    Primary ultrasound end point measurement will be obtained by: 
 
 
 
B-mode ultrasonograms in the posterior view for intima-media thickness (CIMT)   
 
 
measurements of the distal centimeter of the right common carotid artery far wall. 
 
2)    Co-primary ultrasound end point measures will be obtained by: 
 
B-mode ultrasonograms in the posterior view for average diameter measurements during 
systole and diastole of the distal centimeter of the right common carotid artery lumen. The 
luminal dimensions will be used to calculate the following stiffness measurements: 
 
 
 
 
 
 
a) Arterial distensibility = [2(Ds-Dd)/Dd]/PP 
 
 
 
 
 
 
b) Arterial compliance = (D2s-D2d)/PP 
 
where, Ds = average diameter in systole; Dd = average diameter in diastole; PP = pulse 
pressure = systolic BP – diastolic BP 

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9.3.1  Data Transmission 
 
Ultrasound measurements will be obtained by the study ultrasonographers. Data will be routinely sent to 
the DCC electronically. SV ultrasounds will be immediately processed for the primary end point 
measurement; SV CIMT data will be sent to the DCC to perform the stratified randomization. 
Remaining ultrasounds will be processed in batch as the participant completes the trial. 
 
9.3.2  Study Data 
 
1)    For each study ultrasound image, the following measures will be obtained: 
 
a) Carotid artery IMT, averaged over the distal centimeter of the right common carotid 
artery far wall (primary trial end point). 
 
b) Carotid artery luminal diameter, averaged over the distal centimeter of the right 
common carotid artery lumen during systole and diastole. Carotid artery diameters will 
be used in conjunction with pulse pressure to calculate the two measures of arterial 
stiffness (co-primary trial end points – arterial distensibility and arterial compliance). 
 
9.3.3  Data Collection Schedule 
 
Study ultrasounds will be obtained at two pre-randomization visits, SV and v00 and every six months 
on-trial (study visits v06, v12, v18, v24, v30 and v36). 
 
9.3.4  Responsibilities of Ultrasound Laboratory 
 
1) 
Follow protocol for ultrasound acquisition and obtain carotid ultrasounds on each participant at 
 
scheduled study visits. 
 
2) 
Follow protocol for analysis of ultrasound images and obtain study measures. 
 
3) 
Regular backup of study data. 
 
4) 
Immediately process SV ultrasounds for right carotid artery IMT measurement and transmit data to 
 
DCC. 
 
5) 
Use data reports from DCC identifying participants who have completed the trial. Process 
completed participant ultrasound data and electronically transmit study data to the DCC. 
 
6) 
Work with the DCC personnel in rectifying data outliers and errors. 
 
9.3.5  DCC Responsibilities 
 
1) 
Routinely send reports of completed participants to ultrasound laboratory. 
 
2) 
Receive data electronically from study ultrasonographers. 
 
3) 
Review new ultrasound data for completeness. This will include identification of any missing 
 
ultrasound records as well as identification of missing/inaccurate data within an ultrasound record. 
 

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4) 
Review new ultrasound data for accuracy. This will include identification of errors in study visit 
 
numbers, dates and possible outliers in ultrasound data. 
 
5) 
Compare participant's data with previous ultrasound data to monitor possible errors in obtaining 
ultrasound images or in analysis of these images. 
 
6) 
Work with study ultrasonographers in rectifying missing/inaccurate data. 
 
7) 
Once new ultrasound data are deemed clean, update master ultrasound database. 
 
9.4  Cognition 
 
9.4.1  Data Transmission 
 
Cognitive measurements will be obtained by the cognitive tester. The cognitive tester will complete the 
cognitive test scores, complete the summary score form and enter the summary scores into the trial 
database. 
 
9.4.2  Study Data 
 
Cognitive function will be determined with a neuropsychological battery designed to evaluate 7 
cognitive domains including: attention, concentration, working memory, executive function; 
visuospatial/visuoconstructive skills; naming/semantic memory; and verbal and nonverbal episodic 
memory. Neuropsychological tests and corresponding cognitive skills are the following: 
 
1) 
Symbol Digit Modalities Test 
2) 
Trail Making Test, Part B 
3) 
Shipley Institute of Living Scale, Abstraction scale 
4) 
Letter-Number Sequencing 
5) 
Block Design 
6) 
Judgment of Line Orientation 
7) 
Animal Naming 
8) 
Boston Naming Test 
9) 
California Verbal Learning Test 
10) East Boston Memory Test 
11) Faces I and II 
12) Benton Visual Retention Test 
13) Stroop Color and Word Test 
14) Wechsler Test of Adult Reading (verbal intelligence quotient) 
 
9.4.3  Data Collection Schedule 
 
Cognitive assessments will be performed at v00 (baseline) and at study visits v18 and v36. 
 
9.4.4  Responsibilities of Cognitive Tester 
 
1) 
Follow protocol for cognitive testing and obtain cognition data on each participant at scheduled 
study visits. 
 
2) 
Follow protocol for analysis and summarization of cognitive data and obtain study measures. 

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3) 
Enter cognitive summary data into trial data system as it is completed. 
 
4) 
Work with the DCC personnel in rectifying data errors. 
 
9.4.5  DCC Responsibilities 
 
1) 
Monitor timeliness of data entry by study cognitive tester. 
 
2) 
Review newly entered cognition data for completeness. This will include identification of any 
missing cognition data as well as identification of missing/inaccurate data within a cognitive 
assessment. 
 
3) 
Review new cognitive data for accuracy. This will include identification of errors in study visit 
 
numbers, dates and possible outliers in cognitive data. 
 
4) 
Work with study cognitive tester in rectifying missing/inaccurate data. 
 
5) 
Once new cognition data are deemed clean, update master cognition database. 
 
9.5  DCC Tasks 
 
9.5.1  Data Entry 
 
Prior to data entry, all forms will be inventoried and undergo a 2-step review (study coordinator and data 
monitor) for completeness and apparent accuracy; incomplete/inaccurate forms are returned to the study 
coordinator for rectification. Key features of the data entry system at the time of data entry will include: 
data entry screens that mimic the data forms, must-enter fields for key variables and range checks. A 
random sample of study data will be double entered by 2 individuals and program-compared to monitor 
data entry error rates and to identify need for remediation of data entry processes. Data from external 
laboratories will be entered directly into the trial data system by personnel at the external laboratories. 
Hard copies of the laboratory data will also be routinely obtained by the DCC to verify the accuracy of 
data entry. 
 
9.5.2  Study Reports 
 
9.5.2.1  Recruitment and Study Progress 
 
For each meeting of study personnel as well as the External Data Safety Monitoring Board (EDSMB), a 
report of recruitment and participant progress throughout the study will be generated. The recruitment 
portion of the report will include numbers of participants prescreened by telephone and reasons for 
ineligibility, numbers of participants screened in the ARU research clinic and reasons for ineligibility 
and numbers of participants randomized. The study progress portion of the report will include numbers 
of participants who have completed various phases of the study up to the date of the report, numbers of 
study dropouts and reasons for dropouts. 
 
9.5.2.2  Ideal Clinic Visit Schedule 
 
As each participant is randomized, a schedule of "ideal" visit dates and visit windows will be generated 
by the trial data system. An ideal visit date is defined to be the exact date on which the clinic 
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visit windows will be computed as the ideal visit date, plus or minus fourteen (14) days. 
 
Each participant's ideal visit schedule will be generated at randomization. The ideal schedule will be 
available to clinic personnel through the trial data system, with an option to print the schedule as needed. 
The ideal visit schedule will be used to monitor missed visits and compliance to visit schedule (in/out of 
window visits). 
 
9.5.2.3  Data Quality 
 
The data quality report will be generated on a monthly basis and for the EDSMB, and will include data 
on the numbers of errors in completion of study forms. Inaccuracies as well as incompleteness in data 
forms will be considered errors and will be reported separately. Errors will be computed by study form 
and will be presented since the last report (i.e., in the past month) and on a cumulative basis. 
 
The data quality report will also include reports of data queries, including number of queries responded 
to, timeliness of response and number of outstanding data queries. 
 
This report will also include summarization of missed visits and visits outside of the ideal visit window. 
 
9.5.2.4  Participant Safety and Adverse Events 
 
For the EDSMB, the DCC will generate a safety report enumerating the following events, both on a 
cumulative basis (from the start of the study to present) and new events in the past year. For the 
EDSMB, adverse events will be presented by blinded study group. 
 
 1)  Laboratory chemistry values out of range (enumerating abnormally low and high values 
separately). This will be separately reported for first time versus repeated abnormalities. 
 
 2)  Triglycerides >500 mg/dL. First time versus repeated elevations will be separately reported. 
 
 3)  Out of range coagulation and hemostatic factors, PT, aPTT, fibrinogen and platelet count 
(enumerating abnormally low and high values separately). This will be separately reported for first 
time versus repeated abnormalities. 
 
 4)  Abnormal bleeding events ascertained from the Bleeding Assessment Questionnaire [FORMS 6a, 
6b]. 
 
 5)  Abnormal urine dipstick. 
 
 6)  Major cardiovascular events:  fatal/nonfatal MI, sudden coronary death, hospitalization for 
unstable angina, and revascularization procedures (CABG and PTCA). 
  
 7)  Major cerebrovascular events:  TIA, RIND, CVA. 
 
 8)  Peripheral vascular events including surgical reconstruction and bypass. 
 
 9)  Deep vein thrombosis 
 
10)  Pulmonary thromboembolism 
 

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11)  Cancers 
 
12)  Deaths 
 
13)  Other adverse events; serious and non-serious 
 
9.5.2.5  Screening and On-Trial Lipid Levels 
 
As each participant completes all screening visits, clinic personnel will use the trial data system to 
generate a report of lipid levels. On-trial LDL-C levels will also be generated as needed from the trial 
data system by clinic personnel. 
 
9.5.2.6  Participant Compliance 
 
For each participant, compliance to the study products will be monitored and summarized in a monthly 
report. Non-compliance will be reported to the principal investigator. Compliance will be assessed by 
the following: 
 
1) 
Compliance to active nattokinase and placebo nattokinase will be assessed by capsule count. 
Compliance will be defined as >80% of study product consumed. Capsule count will be the 
primary determinant of compliance. 
 
2) 
Overall study compliance will be determined by number of missed study visits. 
 
9.5.3  Data Management 
 
9.5.3.1  Study Databases 
 
The following data tables will comprise the study data: 
 
 1) 
Telephone screening 
 
 2) 
Demographic information 
 
 3) 
Cardiovascular disease history 
 
 4) 
Bleeding assessment questionnaire 
  
 5) 
Active/passive smoking history 
 
 6) 
Alcohol and other drug history 
 
 7) 
Reproductive history (female) 
 
 8) 
Medical history 
 
 9) 
Rose questionnaire 
 
10) Claudication questionnaire 
 

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11) Cardiovascular disease risk factors 
 
12) Non-study medications 
 
13) Nutraceuticals and supplements 
 
14) Vital signs (blood pressure, pulse rate); weight and height 
 
15) Final eligibility checklist 
 
16) Dietary food frequency 
 
17) Nutritional supplements 
 
18) Soy Food Questionnaire 
 
19) Physical activity 
 
20) CES-D Scale 
 
21) Cognitive assessment data 
 
22) Waist:hip ratio 
 
23) ECG 
 
24) Study product compliance 
 
25) Termination/Dropout 
 
26) Carotid ultrasound data 
 
27) Closeout 
 
28) Adverse events including: on-trial cardiovascular, cerebrovascular, peripheral vascular events; on-
trial non-vascular events; on-trial deaths. 
 
29) Quest data: chemistry panel, insulin, complete blood count, platelet count, prothrombin time, 
activated partial thromboplastin time, fibrinogen 
 
30) Hematology laboratory data: coagulation factors (vWF antigen, tPA, PA-1, D-dimers, platelet 
aggregation (ADT, Collagen, Epinephrine), ristocetin cofactor, factor VIII 
 
31) Nattokinase plasma levels, anti-nattokinase antibodies 
 
32) Rheology laboratory data: erythrocyte sdimentation rate, red blood cell aggregation (plasma), red 
blood cell aggregability (polymer), plasma viscosity, whole blood viscosity. 
 
 
 

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33) Biochemistry laboratory data: plasma inflammatory markers (MCP-1, IL-8, TNFα, IL-1β, IL-10, 
E-selectin, P-selectin), endothelial injury in-vitro assay inflammatory markers (VCAM-1, ICAM-
1) and monocyte activation (FACS) markers (CD11b, CD11c, VLA-4) 
 
34) ARU Core Lipid Laboratory data: lipids, C-reactive protein, hemoglobin A1c 
 
35) Sample and buffy coat storage inventory 
 
36) ApoE genotype 
 
9.5.3.2  Data Dictionary 
 
The DCC will maintain a data dictionary that contains the following: 
 
1) 
Copies of all study forms. This will include a historical accounting of all study forms. Thus, all 
 
versions of a study form that are used in the study will be kept with the date of use of each version 
 
indicated on that form. 
 
2) 
A notated copy of each study form that lists the trial database on which these data are entered and 
 
the variable names used for each variable on the form. 
 
3) 
Coding lists that will detail the variable name (and associated forms) for which the coding is used. 
 
4) 
A complete listing of all study variables that includes the following information: 
 
a) Variable name (database system name) 
  
b) Variable description 
 
c) Form from which the variable is derived 
 
d) Data table in which the variable is entered 
 
e) Notation of relevant variable codes (or reference to the appropriate coding list) 
 
9.5.3.3  Quality Control 
 
Quality control of the data will include the following: 
 
1) 
Clinical databases: 
 
a) Clinic coordinator to review all study forms for completeness and accuracy prior to  
 
  
  
sending to DCC. 
 
b) Study programmers and data clerk to review all data forms for completeness and  
 
  
  
accuracy prior to data entry. 
 
c) Run error check routines on newly entered data. These programs will identify logical  
 
  
  
errors in the data, missing data, out-of-range or implausible values, and data outliers  
 
  
  
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d) Based on b and c above, generate data queries in the trial data system to the appropriate 
study personnel. 
 
e) Monitor resolution and completeness of responses to data queries. 
 
f) Edit databases. 
 
2) 
Laboratory and end point data: 
 
a) Monitor measures for any apparent longitudinal data drift. 
 
b) Monitor timeliness of receipt of laboratory data and end point data and identify   
 
  
  
outstanding laboratory samples. 
 
c) Perform error check programs and data queries as in section 1 above. 
 
d) Edit databases. 
 
3) 
Generate routine quality control reports that will include reporting (by study month) of the 
numbers of errors in completion of study forms, by form, as well as timeliness and completeness 
of response to data queries. 
 
4) 
Monitor study for protocol violations. 
 
5) 
Double entry of random key study data for monitoring of key stroke error rate. 
 
6) 
Biannual audit of random data sample for comparison of hard copies to complementary databases. 
 
7) 
Inform Data Safety Monitoring Board and principal investigator of ongoing/repeated irregularities 
 
in any study data. 
 
9.5.3.4  Backup of Databases 
 
All study databases will be backed up to secure servers on a daily basis. Each data file will also be 
copied to an external hard drive on a weekly basis and stored off-campus. External hard drive copies of 
the data base will capture all versions of the data base for the previous 4 weeks. 
 
9.6  Statistical Analysis of Study Data 
 
9.6.1  Preliminary Analyses 
 
Standard procedures, including frequency distributions and histograms, summary statistics (means, 
medians, variances, ranges), and EDA procedures (box plots, stem and leaf displays), will be used to 
describe the distributions of data, determine normalizing transformations (if necessary), and identify 
data outliers. These analyses will be conducted for all study outcome measures as well as study 
covariates.  If continuous outcome variables are not normally distributed, normalizing transformations 
will be considered so that parametric analytic methods may be used; non-parametric methods may be 
used as an alternative. 
 
 

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9.6.2  Baseline Comparability 
 
All participants will be compared between treatment arms with respect to demographic (e.g., age, race, 
gender), cardiac and other medical history factors, cardiovascular risk factors (including blood pressure, 
smoking status), dietary factors, baseline CIMT, vascular stiffness, cognition, plasma insulin, 
lipids/CRP, hemoglobin A1c, coagulation factors, rheology parameters and inflammatory variables. 
Analytical methods will include two-sample t-tests (or a non-parametric analog) for continuous variables 
and chi-square procedures for categorical variables. The significance level for all analyses will be set at 
a two-sided α=0.05. 
 
If multiple baseline laboratory measures are completed (e.g., at SV and at v00), the baseline value will 
be computed as the average of the screening measure and the v00 visit determined prior to intervention. 
All treatment group comparisons on baseline variables will be performed for the entire randomized 
study sample, for the group of participants with repeat ultrasounds, and for the group of participants 
without repeat ultrasounds. 
 
9.6.3  Comparison of On-trial Changes in Laboratory and Lifestyle Variables 
 
Treatment groups will be compared with respect to on-trial levels and on-trial changes in plasma insulin, 
lipids/CRP, hemoglobin A1c, coagulation factors, rheology parameters and inflammatory variables. 
Study groups will also be compared for on-trial levels and on-trial changes of other factors that might 
confound the analysis of primary end point differences. These will include analysis of dietary factors 
(e.g., total calories, and total calorie-adjusted calories from total, saturated, polyunsaturated, and 
monounsaturated fats, calories from protein, calories from carbohydrates, and dietary genistein, 
daidzein, and glycitein), physical activity, cardiovascular risk factors (including blood pressure, smoking 
status), and use of relevant nonstudy medications (including antihypertensive and lipid-lowering 
medications). For descriptive purposes, on-trial laboratory, dietary, clinical, and physical activity 
variables will be computed as the average of all on-trial determinations, weighted by the length of time 
between determinations. On-trial smoking will be compared as percentage of smokers versus percentage 
abstainers; average on-trial smoking levels (cigarettes per day) will also be computed for smokers as the 
average of all on-trial determinations as above. For all laboratory and clinical measures, a change 
variable will be computed at each on-trial measurement time as: on-trial value minus baseline value.  
Changes in laboratory and clinical variables from baseline will be compared within each treatment arm 
using generalized estimating equations to account for the repeated measurements over the study period; 
the model-specified correlation structure of the repeated measures will be determined by examination of 
the observed correlation structure. The test that the model intercept differs from 0 will comprise the 
overall test for significant change from baseline; incorporation of covariates for time periods of the 
study will test for differential change from baseline over the study period. Comparison of on-trial levels 
and changes in laboratory and clinical variables across treatment groups will also use generalized 
estimating equations, with an added covariate for randomized treatment group; interaction terms of 
treatment group-by-study time period will test whether treatment group differences vary over the study 
period. Compliance with the study product will be compared across treatment groups, also using 
generalized estimating equations as described. All analyses will be performed for the entire study 
sample and for the group of participants whose overall study product compliance is >80%. The 
significance level for all analyses will be set at a two-sided α=0.05. 
 
 
 
 
 

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9.6.4  Evaluation of Potential Biases and Study Validity Introduced by Differential Dropout 
 
Possible biases introduced by differential study dropout will be evaluated by comparing: 
 
1) 
Baseline variables (Section 9.6.2) in participants with and without repeat ultrasounds, and in 
participants with and without repeat cognitive testing. Analyses will be performed by two-sample 
t-tests, Wilcoxon rank sum, or chi-square procedures as appropriate. 
 
2) 
On-trial variables (Section 9.6.3) evaluated within treatment group in participants with and without 
repeat ultrasounds, and in participants with and without repeat cognitive testing. Analyses will be 
performed by analysis of variance (factors for repeat ultrasound/cognitive test (yes/no) and 
treatment group) and Mantel-Haenszel chi-square (with stratification on treatment group) 
procedures. 
 
3) 
Participants without repeat ultrasounds (or cognitive testing) by treatment group on baseline and 
on-trial variables (Sections 9.6.2 and 9.6.3). Analyses will be performed by two-sample t-test, 
Wilcoxon rank sum or chi-square procedures as appropriate. 
 
9.6.5  Analysis of Study Outcomes 
 
All analyses of study outcomes will be conducted on an intent-to-treat basis. All participants with 
baseline and any follow-up end point data, regardless of their dropout and compliance status, will be 
analyzed according to their randomized treatment group. Statistical assumptions of the planned analyses 
will be verified. Alternative analyses will be performed if the assumptions of the planned analyses are 
not justified. Key assumptions that will be verified are: 1) normality of primary end point; 2) 
homogeneity of variances between treatment groups; and, 3) deviations from linearity in the regression 
of end point measurements (CIMT, arterial stiffness, cognition) against time on-study. 
 
9.6.5.1  Primary Efficacy Analysis:  Carotid Artery IMT Analyses 
 
The primary end point will be the per-subject rate of change in the right distal CCA far wall IMT. The 
rate of change in the right distal CCA far wall CIMT will be computed for each participant by fitting a 
regression line of CIMT on years since baseline ultrasound. The estimated slope of the regression line 
will be used as that subject's CIMT rate of change. Analyses of trial end points will be performed on the 
intent-to-treat sample, which will be defined as all randomized participants who had a baseline and at 
least 1 follow-up CIMT measurement taken after randomization. 
 
The statistical approach outlined above will be accomplished using mixed effects models for 
longitudinal data, with the randomization stratification variable (dichotomized baseline CIMT) used as a 
covariate. Each CIMT measure will be included as a dependent variable. The regression coefficient 
associated with the model covariate of time on-study will estimate the average group progression rate of 
CIMT (in mm/year). Random effects terms for the regression intercept and slope (change over time) will 
allow for individual participant deviations from their group average in baseline CIMT and CIMT rate of 
change, respectively. The statistical test for differences in the CIMT rate by treatment group will be 
accomplished by adding a treatment group by time on-study interaction term. The significance of the 
regression coefficient associated with this interaction term is the test that the CIMT rates differ by 
treatment group. We will also evaluate baseline plasma levels of insulin, lipids/CRP, hemoglobin A1c, 
coagulation factors, rheology parameters and inflammatory variables, dietary nutrients, smoking, blood 
pressure, physical activity and use of non-study medications as potential confounders. If any of these 
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covariates in the analysis. 
 
9.6.5.2  Co-Primary Efficacy Analysis:  Carotid Artery Stiffness Analyses 
 
Statistical methods to evaluate the arterial stiffness outcomes will mirror those specified for the CIMT 
analyses above. A normalizing transformation will be applied to the arterial stiffness measures prior to 
analysis if required. 
 
9.6.5.3  Secondary Efficacy Analysis:  Cognition Analyses 
 
Three composite cognitive measures will be used to test for randomized treatment group differences in 
cognition; each composite will be considered as co-secondary endpoints. (1) The global composite will 
use scores from all neuropsychological tests. (2) The verbal memory composite will use the California 
Verbal Learning Test and East Boston Memory Test immediate and delayed recall scores. (3) The 
executive function composite (determined from principal components of prior trial data) will use scores 
from the Symbol Digit Modalities Test, the Trail Making Test, the Shipley Abstraction scale, Letter-
Number Sequencing, and category fluency. The composites will be calculated as the average of 
component standardized scores weighted by the inverse interest correlation matrix. Absolute values of 
change from baseline on each cognitive endpoint will be calculated at both post-randomization cognitive 
follow-ups (18 and 36 months). General estimating equation models will be used to analyze the repeated 
measures of change in the cognitive endpoints. The primary independent variables will be randomized 
treatment group; covariates will include the CIMT randomization stratification variable as well as the 
baseline value of the cognitive endpoint. Differences in cognitive change will be tested by treatment 
group. Further analyses will test for interactions with test time (18 versus 36 months) to evaluate 
whether treatment effects (treatment by time interaction) vary by follow-up time. 
 
9.6.5.4  Analysis of Clinical Events, Adverse Experiences, and Safety Measures 
 
The following clinical events will be tabulated and compared between treatment groups using chi-square 
or Fisher's exact methods: 
 
1) 
Major cardiovascular events:  fatal/nonfatal MI, sudden coronary death, hospitalization for 
unstable angina, and revascularization procedures (CABG and PTCA). 
  
2) 
Major cerebrovascular events:  TIA, RIND, CVA. 
 
3) 
Deep vein thrombosis. 
 
4) 
Pulmonary thromboembolism. 
 
5) 
Incident cancers. 
 
6) 
All cause mortality and cause-specific mortality, including cardiovascular, cerebrovascular, 
 
peripheral vascular, cancer, other medical, and non-medical and non-coronary mortality. 
 
7) 
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In the analysis of laboratory safety variables, treatment groups will be compared with regard to 
proportions of participants with clinically meaningful changes (either increase or decreases from normal 
range). Analyses of these data will be done using a chi-square test or Mantel-Haenszel test that allows 
for stratification, as appropriate. 
 
9.6.6  Interim Analysis 
 
At each meeting of the EDSMB, summary tables of safety data (Section 9.5.2.4) without any formal 
statistical analyses will be reviewed. The EDSMB will be masked to treatment assignment (i.e., the 
treatments will be labeled as A and B). If in the best judgment of the EDSMB, trends or patterns are 
observed in the safety data, the EDSMB may request a formal statistical analysis of the data. The Lan-
DeMets rule to determine the current (and all future) alpha levels will be used. Early termination is 
predicated on attaining statistical significance on major safety issues to be defined by the EDSMB. 
 
Interim analyses will not be performed on the primary (CIMT, arterial stiffness) and secondary 
(cognition) trial outcomes. 
 
9.6.7  Ancillary Statistical Analyses 
 
9.6.7.1  Within-Group Analyses 
 
The effects of on-trial end point variables other than treatment modality on the per-subject rate of 
change in CIMT, arterial stiffness and cognition will be separately assessed. The parameters to be tested 
will include laboratory data (plasma insulin, lipids/CRP, hemoglobin A1c, coagulation factors, rheology 
parameters, inflammatory variables and nattokinase levels), clinical variables (blood pressure, body 
mass, waist:hip ratio, etc.), and lifestyle variables (dietary intake, physical activity, smoking, etc.). 
Baseline and on-trial values of these data will be collected in order to address questions of clinical 
significance within each treatment group such as: 1) which baseline characteristics have differential 
effectiveness on the rate of change in CIMT; and, 2) what is the role of changes in these variables on the 
rate of change in CIMT. These analyses will utilize mixed effects models and generalizing estimating 
equations with CIMT (arterial stiffness, cognition) as the dependent variable. Analyses will be 
conducted in the combined sample with a covariate included to control for treatment group. Interaction 
terms with treatment group will be introduced to test whether relationships between independent 
variables and the trial end point are of equal magnitude between the 2 study groups. Experiment-wise 
error rates in these analyses will be controlled. All participants contributing at least one follow-up 
ultrasound (or cognitive test) will be included in these analyses. 
 
9.6.7.2  Modifiers of Treatment Efficacy 
 
The extent to which treatment effects are modified by other subject-specific factors will be examined.  
Potential treatment effect modifiers include baseline CIMT, smoking status (on-trial smokers versus 
non-smokers), BMI, age and gender. The statistical significance of differences in the treatment effect by 
subgroup will be tested by inclusion of an interaction term (treatment by subgroup) in the efficacy 
models detailed above. For descriptive comparisons, treatment effect sizes will be computed for each 
level of the possible modifier. 
 
9.6.7.3  Mediators of Treatment Efficacy 
 
We will test the hypothesis that nattokinase treatment-related reductions in atherosclerosis and cognitive 
measures will be mediated through hemostatic, fibrinolytic and hemorheological factors as well as 
attenuation of inflammation, monocyte activation, vascular endothelium injury and activation of 

 
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vascular endothelium by circulating monocytes. We will use structural equation models to test for 
mediation.  Observed outcome variables will be changes in CIMT (annualized change rate), stiffness 
(annualized change rate) or cognitive composite scores. For each of the potential mediator that shows a 
significant treatment effect, we will test the direct effects of randomized treatment and the indirect 
effects of treatment (i.e., treatment acting through the potential mediators).  Bootstrapping will be used 
to obtain 95% confidence intervals on the direct and indirect effects; confidence intervals that exclude 
the value of 0 will be considered as evidence for statistical significance.  
 
9.6.7.4  Correlations in Atherosclerosis and Cognitive Outcome Measures 
 
We will use mixed effects models to test the hypothesis that the reduction in subclinical atherosclerosis 
progression and cognitive decline with nattokinase will be correlated. The dependent variable will be 
each value of the CIMT (or arterial stiffness) measure at each study time point. The primary independent 
variables will be the 18-month and 36-month change in cognitive composites. Interaction terms of time 
on study with the cognitive composite change scores will test whether the rate of change in CIMT (or 
arterial stiffness) is associated with the change in cognition.