RoBotic TCD Ultrasound BubbLe Study Compared to Transthoracic Echocardiography for Detection of Right to Left Shunt
Phase: NA
Conditions: Embolic Stroke of Undetermined Source, Transient Ischemic Attack, Right-To-Left Atrial Shunt, Patent Foramen Ovale
Sponsor: NovaSignal Corp.

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CONFIDENTIAL 
RoBotic TCD Ultrasound BubbLe Study Compared to Transthoracic 
Echocardiography for Detection of Right to Left Shunt 
The BUBL Study 
Date: 
02 AUG 2021 
Version: 
05 
Protocol No.: 
NA-07BBL-01 
Sponsor: 
NovaSignal Corp. 
2440 S. Sepulveda Blvd., Suite 115 
Los Angeles, CA 90064  
Revision History: 
Version Date 
Description 
Version 01 – 21MAY2020 
Original Document – not implemented 
Version 02 – 15JUN2020 
Amendment to add exclusion criteria #2 for 
pregnant women per WIRB recommendation 
Version 03 – 11AUG2020 
Administrative change from Neural Analytics 
to NovaSignal company name change 
Version 04 – 12FEB2021 
Removed upper limit of age Inclusion Criteria. 
Added continuation of enrollment after the 
first 150 subjects with TTE are enrolled up to 
another 150 subjects with TEE performed 
Version 05 – 02AUG2021 
Revised Sub-Study to include TTE or TEE 
after Main Study 150 subjects are enrolled. 
Removed single-site sub-study at UTHSC. 
Confidentiality Statement 
The information contained in this document, especially unpublished data, is confidential and the 
proprietary property of NovaSignal. It is therefore provided to you in confidence as an investigator, 
potential investigator, or consultant, for review by you, your staff, and if necessary, an Institutional 
Review Board. It is understood that this information will not be disclosed to others without written 
authorization from NovaSignal except to the extent necessary to obtain informed consent from 
those persons who may receive the medical device. 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 2 of 44 
Protocol Signature Page- Principal Investigator 
 
I have read the protocol entitled, Robotic TCD Ultrasound Bubble Study Compared to 
Transthoracic Echocardiography for Detection of Right to Left Shunt, and agree that it contains all 
necessary details for carrying out the described study. I will conduct this study as outlined therein 
and will make reasonable effort to complete the study within the designed time frame. I will provide 
copies of the study plan and all information furnished by NovaSignal to all study personnel under 
my supervision. I will discuss this material with them to assure that they are fully informed about 
the device and the conduct of this study. 
 
I will ensure that the study is conducted according to applicable regulations, to applicable laws 
and to hospital policy and Institutional Review Board (IRB) and/or Ethics Committee (EC) 
requirements. 
 
 
 
 
_______________________ 
________________________ _______/________/________ 
Principal Investigator’s  
Principal Investigator’s 
Date (dd/mmm/yyyy) 
Printed Name 
Signature 
 
 
 
 
 
 
 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 3 of 44 
Protocol Signature Page- Sub-Investigator 
 
I have read the Study entitled, Robotic TCD Ultrasound Bubble Study Compared to Transthoracic 
Echocardiography for Detection of Right to Left Shunt, and agree that it contains all necessary 
details for carrying out the described study.  
 
I will ensure that the study is conducted according to applicable regulations, to applicable laws 
and to hospital policy and Institutional Review Board (IRB) and/or Ethics Committee (EC) 
requirements. 
 
 
 
 
_______________________ 
________________________ ________/_______/________ 
Sub- Investigator’s  
Sub-Investigator’s 
Date (dd/mmm/yyyy) 
Printed Name 
Signature 
 
 
 
 
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
DWF-C0001-001
CONFIDENTIAL
Page 4 of 44
TABLE OF CONTENTS
PROTOCOL SYNOPSIS ..................................................................................................................................... 7
LIST OF ABBREVIATIONS......................................................................................................................................13
BACKGROUND AND RATIONALE OF STUDY................................................................................................... 14
BACKGROUND INFORMATION ..............................................................................................................................14
CURRENT OPTIONS............................................................................................................................................14
LIMITATIONS ....................................................................................................................................................14
RATIONALE ......................................................................................................................................................15
POTENTIAL RISKS AND BENEFITS...........................................................................................................................15
DEVICE DESCRIPTION.................................................................................................................................... 15
LUCID M1 TRANSCRANIAL DOPPLER SYSTEM..........................................................................................................15
NEURALBOT INVESTIGATIONAL SYSTEM (NB-IS).....................................................................................................16
INTENDED USE OF THE DEVICE..............................................................................................................................16
CONTRAINDICATIONS.........................................................................................................................................17
STUDY DEVICE TRAINING REQUIREMENTS..............................................................................................................17
DEVICE STORAGE AND ACCOUNTABILITY................................................................................................................18
STUDY OBJECTIVES ....................................................................................................................................... 18
PRIMARY EFFICACY ENDPOINT .............................................................................................................................18
SECONDARY TECHNICAL EFFICACY ENDPOINTS: .......................................................................................................18
EXPLORATORY ENDPOINTS: ................................................................................................................................. 18
PRIMARY SAFETY ENDPOINT: ...............................................................................................................................18
SECONDARY SAFETY ENDPOINTS: .........................................................................................................................18
STUDY DESIGN.............................................................................................................................................. 19
STUDY POPULATION SIZE....................................................................................................................................19
STUDY DURATION .............................................................................................................................................19
STUDY ENTRY CRITERIA – TTE MAIN STUDY...........................................................................................................19
5.3.1
Inclusion Criteria...................................................................................................................................19
5.3.2
Exclusion Criteria..................................................................................................................................19
STUDY ENTRY CRITERIA – TTE OR TEE SUB-STUDY (AFTER FIRST 150 SUBJECTS WITH TTE ENROLLED) ..............................20
5.4.1
Inclusion Criteria...................................................................................................................................20
5.4.2
Exclusion Criteria..................................................................................................................................20
STUDY PROCEDURES..................................................................................................................................... 20
INFORMED CONSENT AND SCREENING...................................................................................................................20
NUMBERING OF STUDY SUBJECTS.........................................................................................................................21
SCREEN FAILURES..............................................................................................................................................21
STUDY DISCONTINUATION BY IRB OR SPONSOR ......................................................................................................21
STUDY SCHEDULE..............................................................................................................................................22
6.5.1
Schedule of Assessments......................................................................................................................22
SCANNING/DATA ACQUISITION............................................................................................................................22
NEURALBOT INVESTIGATIONAL SYSTEM (NB-IS) SCANNING TECHNIQUE......................................................................22
ALARA CONSIDERATIONS...................................................................................................................................23
STANDARD OF CARE PROCEDURES........................................................................................................................24
STUDY-SPECIFIC PROCEDURES .............................................................................................................................24
SCREENING PROCEDURES....................................................................................................................................24
PROCEDURE –NB-IS TCD SCANNING SESSION........................................................................................................24
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
DWF-C0001-001
CONFIDENTIAL
Page 5 of 44
6.12.1
Patient Preparation – Valsalva Maneuver (VM) ..................................................................................24
6.12.2
Contrast Agent Preparation .................................................................................................................24
6.12.3
SOC Transcranial Doppler Ultrasound Procedure (TCD) (as applicable)...............................................25
6.12.4
NeuralBot Investigational System (NB-IS) TCD Procedure....................................................................25
6.12.5
Transthoracic Echocardiography Procedure (TTE) per SOC..................................................................26
6.12.6
Transesophageal Echocardiography Procedure (TEE) per SOC (as applicable)....................................26
6.12.7
Follow-Up Procedures ..........................................................................................................................27
NB-IS TCD DATA COLLECTION AND DATA TRANSFER ................................................................................................27
STUDY WORKFLOW............................................................................................................................................28
RISKS AND BENEFITS..................................................................................................................................... 29
KNOWN POTENTIAL RISKS...................................................................................................................................29
BENEFITS.........................................................................................................................................................29
SAFETY ASSESSMENTS .................................................................................................................................. 29
SAFETY PARAMETERS.........................................................................................................................................29
8.1.1
Adverse Event (AE) ...............................................................................................................................29
8.1.2
Serious Adverse Event (SAE).................................................................................................................30
8.1.3
Unanticipated Adverse Device Effect (UADE/USADE) ..........................................................................30
8.1.4
Device Deficiency, Device Malfunction, and Use Error.........................................................................30
REPORTING DEVICE AND/OR PROCEDURE RELATED ADVERSE EVENTS..........................................................................31
CLASSIFICATION OF A DEVICE AND/OR PROCEDURE RELATED ADVERSE EVENT...............................................................32
8.3.1
Severity Rating of Event .......................................................................................................................32
8.3.2
Relationship of Study Device and/or Procedure...................................................................................32
8.3.3
Outcome of Event................................................................................................................................. 32
REPORTING PROCEDURES ...................................................................................................................................33
PROTOCOL DEVIATIONS ............................................................................................................................... 33
QUALITY CONTROL AND QUALITY ASSURANCE......................................................................................... 34
ORGANIZATIONAL PREPARATIONS ........................................................................................................................34
TRAINING ........................................................................................................................................................34
DATA QUALITY ASSURANCE................................................................................................................................. 34
SUBJECT PRIVACY..............................................................................................................................................34
CASE REPORT FORMS.........................................................................................................................................35
INVESTIGATIONAL DEVICE ACCOUNTABILITY............................................................................................................35
SELECTION OF INVESTIGATORS .............................................................................................................................35
CLOSE-OUT DOCUMENT REVIEW..........................................................................................................................35
STATISTICAL ANALYSIS.............................................................................................................................. 35
PRIMARY ENDPOINT ANALYSIS.............................................................................................................................35
SECONDARY TECHNICAL EFFICACY ENDPOINT(S) ANALYSIS ........................................................................................36
EXPLORATORY ENDPOINTS ANALYSIS.....................................................................................................................36
IMAGING CORE LAB ANALYSIS .............................................................................................................................37
RLS/PFO VALIDATION AND ANALYSIS...................................................................................................................37
SAMPLE SIZE CALCULATION................................................................................................................................. 37
PRIMARY SAFETY ENDPOINT ANALYSIS ..................................................................................................................39
SECONDARY SAFETY ENDPOINT ANALYSIS ..............................................................................................................39
ETHICAL CONSIDERATIONS ....................................................................................................................... 39
INVESTIGATIONAL REVIEW BOARD (IRB) APPROVAL ................................................................................................39
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
DWF-C0001-001
CONFIDENTIAL
Page 6 of 44
ROLE OF NOVASIGNAL .......................................................................................................................................39
INVESTIGATOR RESPONSIBILITIES..........................................................................................................................40
RECORDS CUSTODY ...........................................................................................................................................40
APPENDIX ................................................................................................................................................. 41
SPENCER LOGARITHMIC SCALE (SLS)10..................................................................................................................41
INTERNATIONAL CONSENSUS CRITERIA (ICC)11 .......................................................................................................41
CLASSIFICATION OF POTENTIAL CAUSATIVE MECHANISM IN PFO–ASSOCIATED STROKE1.................................................41
REFERENCES.............................................................................................................................................. 42
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
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CONFIDENTIAL
Page 7 of 44
PROTOCOL SYNOPSIS
Protocol Synopsis:
Study Sponsor:
NovaSignal Corp.
2440 S. Sepulveda Blvd., Ste. 115
Los Angeles, CA 90064
Protocol No.: NA-07BBL-01
Regulatory Class: II
Study Device(s): NeuralBot Investigational
System (NB-IS)
Development Phase: Pivotal
Study Title:
Robotic TCD Ultrasound Bubble Study Compared to Transthoracic Echocardiography for 
Detection of Right to Left Shunt
Overall Study Design:
This study is a multi-center, prospective, single-arm, non-significant risk (NSR) device study in 
which up to 150 evaluable subjects with embolic stroke of undetermined source (ESUS) 
according to standardized criteria13 will be evaluated with NB-IS TCD and standard of care TTE 
to screen for right to left shunt (RLS) or patent foramen ovale (PFO). Additionally, up to 150 
evaluable subjects will be evaluated  with NB-IS TCD and standard of care TTE or TEE.
Objective:
To evaluate the shunt detection rate of the NeuralBot Investigational System (NB-IS) TCD 
relative to standard of care diagnostic techniques (transthoracic echocardiography (TTE), 
transesophageal echocardiography (TEE), and standard transcranial Doppler ultrasound (TCD)
and to assess the safety, accuracy and usability of the NB-IS device.
Number of Subjects: 
TTE Main Study: Up to 150 subjects with 
suspected RLS/PFO receiving TTE
TEE Sub-Study: Up to 150 subjects with 
suspected RLS/PFO receiving TTE or TEE
Number of Sites:
Up to 7 centers total (US)
Duration of Study Participation:
•
Enrollment: approximately 9-15 months
•
Participant duration: 1-3 days (screening, NB-IS TCD, TCD, TTE, TEE); 1-60 days 
(follow-up)
Primary Efficacy Endpoint: 
•
% detection of RLS/PFO with NB-IS TCD compared against standard of care Transthoracic 
Echocardiography (TTE)
Secondary Technical Efficacy Endpoint:
•
% agreement for detection of RLS/PFO with NB-IS TCD vs SOC TCD
•
% agreement for detection of RLS/PFO with
NB-IS
TCD vs Transesophageal 
Echocardiography (TEE)
•
% agreement for detection of intervenable shunts with NB-IS TCD vs TTE
•
No window rate (including both unilateral and bilateral absent acoustic windows)
•
Success rate of the NB-IS TCD
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 8 of 44 
Protocol Synopsis: 
Study Sponsor: 
NovaSignal Corp. 
2440 S. Sepulveda Blvd., Ste. 115 
Los Angeles, CA 90064 
Protocol No.: NA-07BBL-01 
Regulatory Class: II 
Study Device(s): NeuralBot Investigational 
System (NB-IS) 
Development Phase: Pivotal 
• 
Incidence of device malfunctions 
 
Exploratory Endpoints: 
• 
Development of an automated algorithm for Spencer Scale and ICC grading 
• 
Single site sub-study (UTHSC) of right atrial monitoring for timing of Valsalva during NB-IS 
and standard TCD, comparing shunt detection rate with/without right atrial (RA) monitoring 
against one another and each against TEE.  
 
Primary Safety Endpoint: 
• 
Incidence of device-related serious adverse events. 
Entry Criteria TTE Main Study: 
Inclusion Criteria 
1. Subject 18 years of age and older. 
2. Subject presents with a clinical condition characterized by neurological signs and 
symptoms that, in the opinion of the investigator, include embolic stroke or TIA in the 
differential diagnosis. 
3. Scheduled for a transthoracic echocardiograph (TTE) study with agitated saline contrast 
(bubble study) per standard of care within ±30 days of informed consent. 
4. Subject is able to successfully perform a Valsalva Maneuver (VM). 
5. Subject or Legally Authorized Representative has the ability to provide informed consent 
and comply with the protocol.  
Exclusion Criteria 
1. Subject has undergone a right to left shunt (RLS) or patent foramen ovale (PFO) closure. 
2. Female who is pregnant or lactating at time of admission 
3. Subjects who underwent partial or full craniotomy/craniectomy within the past 6 months. 
4. Subjects who have a physical limitation preventing TCD headset placement 
Entry Criteria TTE or TEE Sub-Study (after first 150 subjects with TTE enrolled): 
Inclusion Criteria 
1. Subject 18 years of age and older. 
2. Subject presents with a clinical condition characterized by neurological signs and 
symptoms that, in the opinion of the investigator, include embolic stroke or TIA in the 
differential diagnosis. 
3. Scheduled 
for 
a 
transthoracic 
echocardiograph 
(TTE) 
or 
transesophageal 
echocardiograph (TEE) study with agitated saline contrast (bubble study) per standard 
of care within ±30 days of informed consent. 
4. Subject is able to successfully perform a Valsalva Maneuver (VM). 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 9 of 44 
Protocol Synopsis: 
Study Sponsor: 
NovaSignal Corp. 
2440 S. Sepulveda Blvd., Ste. 115 
Los Angeles, CA 90064 
Protocol No.: NA-07BBL-01 
Regulatory Class: II 
Study Device(s): NeuralBot Investigational 
System (NB-IS) 
Development Phase: Pivotal 
5. Subject or Legally Authorized Representative has the ability to provide informed consent 
and comply with the protocol.  
Exclusion Criteria 
1. Subject has undergone a right to left shunt (RLS) or patent foramen ovale (PFO) closure. 
2. Female who is pregnant or lactating at time of admission 
3. Subjects who underwent partial or full craniotomy/craniectomy within the past 6 months. 
4. Subjects who have a physical limitation preventing TCD headset placement 
Data Collection: 
• 
Informed Consent 
• 
Subject Demographics 
• 
Relevant Medical History 
• 
Physical Exam (SOC) 
• 
NB-IS TCD Operator (name) 
• 
NB-IS TCD Scan information 
o Date/Time of NB-IS TCD bubble study 
o NB-IS TCD scan Start/Stop times with and without VM 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM 
o Head inclination (degree)/subject positioning/location of the exam 
• 
SOC Transcranial Doppler Ultrasound (TCD) local results (SOC), if performed 
o Date/Time of SOC TCD bubble study 
o SOC TCD scan Start/Stop times with and without VM (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
o Head inclination (degree)/subject positioning/location of the exam 
• 
Transthoracic Echocardiography (TTE) local results (SOC) 
o Date/Time of TTE bubble study 
o TTE Start/Stop times with and without VM (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
• 
Transoesophageal Echocardiography (TEE) local results (SOC), if performed 
o Date/Time of TEE exam 
o TEE Start/Stop times (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
• 
Local Spencer Logarithmic Scale (SLS) grades (SOC TCD only) 
• 
Local International Consensus Criteria (ICC) grades (SOC TCD only) 
• 
Classification of Potential Causative Mechanism in PFO–Associated Stroke1 
• 
PFO Closure Decision (post diagnostic procedures) 
• 
PFO Closure Procedure, if performed 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 10 of 44 
Protocol Synopsis: 
Study Sponsor: 
NovaSignal Corp. 
2440 S. Sepulveda Blvd., Ste. 115 
Los Angeles, CA 90064 
Protocol No.: NA-07BBL-01 
Regulatory Class: II 
Study Device(s): NeuralBot Investigational 
System (NB-IS) 
Development Phase: Pivotal 
• 
Relevant Concomitant Medications 
• 
Device and/or Procedure Related Adverse Events (NB-IS and/or NB-IS TCD 
procedure) 
• 
Device Deficiencies / Malfunctions / Technical Observations 
Imaging Core Lab Analysis: 
All imaging data will be sent to a core laboratory which will provide independent quantitative 
and qualitative assessment of all NB-IS TCD and TTE, and SOC TCD and TEE bubble study 
data, if performed. They will be blinded to the study and local diagnostic report data and provide 
independent review. 
 
Data Analysis: 
Evaluation of correlation between diagnostic imaging standard of care (local TCD/TTE/TEE) 
and NB-IS TCD diagnostic performance by an independent core laboratory. Success criteria 
will include clinical validation of NB-IS TCD for RLS/PFO detection targeting (i) 40% increase 
in NB-IS TCD sensitivity compared to TTE sensitivity and (ii) NB-IS TCD sensitivity ≥ 90% 
compared to SOC TCD sensitivity. 
 
 
 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
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CONFIDENTIAL 
Page 11 of 44 
Schedule of Assessments 
Assessments 
Screening1 
Procedure1 
Follow-Up1 
Informed Consent 
X 
 
 
Demographics 
X 
 
 
Relevant Medical History 
X 
 
 
Physical Exam ♦ 
X 
 
 
SOC TCD Bubble Study Exam ♦ 
 
O4 
 
NB-IS TCD Bubble Study Exam 
 
X2 
 
Transthoracic Echocardiograph (TTE) Bubble Study ♦ 
 
X3 
 
Transesophageal Echocardiograph (TEE) ♦ 
 
O4 / X8 
 
PFO Closure Decision ♦ 
 
 
X5 
Classification of Potential Causative Mechanism in PFO–
Associated Stroke7♦ 
 
 
X 
PFO Closure Procedure ♦  
 
 
O6 
Device-Related Adverse Events 
 
X  
 
Procedure-Related Adverse Events 
 
X 
 
Concomitant Medications 
X 
X 
 
Device Deficiencies 
 
X 
 
♦ Standard of Care     X = Required     O = Optional, if performed 
1. 
Screening, Procedure, and Follow-Up Visits can occur on the same day 
2. 
NB-IS TCD performed with contrast agent of 9 ml bacteriostatic saline with 1 ml air mixed 10 times. Performed 
at Rest AND with VM – 60 sec recordings each in supine positions AND with HOB raised to 45 degrees. 
3. 
TTE can be performed within ±30 days of informed consent per SOC. 
4. 
SOC TCD and TEE results collected if performed as applicable per standard of care. 
5. 
PFO closure decision collected post diagnostic imaging procedures. 
6. 
PFO closure procedure data collected within 60 days post diagnostic workup, if performed 
7. 
Elgendy AY, Saver JL, Amin Z, et al; MSCAI14. Proposal for Updated Nomenclature and Classification of 
Potential Causative Mechanism in Patent Foramen Ovale-Associated Stroke. JAMA Neurol. 2020 Apr 13. 
doi:10.1001/jamaneurol.2020.0458. [Epub ahead of print] PubMed PMID: 32282016. 
8. 
SOC TTE or TEE will be required after the first 150 TTE subjects are enrolled and can be performed within ±30 
days of informed consent per SOC. 
 
 
 
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Protocol ID: NA-07BBL-01 
Version Date: August 2, 2021 
 
DWF-C0001-001 
CONFIDENTIAL 
Page 12 of 44 
 
 
Standard of Care 
 
Arrive at Neuro Unit 
Pre-Screened for Enrollment 
1) Scheduled SOC TTE 
2) ≥ 18years of age 
Transesophageal Echocardiograph (TEE) 
As applicable 
NB-IS TCD  
All Clinical Data Points Collected – EMR 
NB-IS TCD/SOC TCD/TTE/TEE Data to  
CORE LAB 
NB-IS TCD Data to NovaSignal 
Subject meet all I/E criteria? 
SOC TCD (as applicable) 
Yes 
No 
SOC TCD/TTE/TEE Results 
PFO Closure Decision &  
PFO-Associated Stroke Classification 
INFORMED CONSENT 
Pre-Screen 
Failure 
Study Procedure 
NB-IS TCD Procedures: 
1. Performed with contrast agent 
of 9 ml saline with 1 ml air  
mixed 10 times. 
2. Performed at Rest AND with 
VM – 60 sec recordings each in 
supine positions AND with HOB 
at 45 degrees (NB-IS & TCD 
only). 
Transthoracic Echocardiograph (TTE) 
PFO Closure Procedure, if performed 
Study Procedure 
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
DWF-C0001-001
CONFIDENTIAL
Page 13 of 44
List of Abbreviations
Abbreviation/Term
Definition
AE
Adverse Event
CBF
Cerebral Blood Flow
CBFV
Cerebral Blood Flow Velocity
CFR
Code of Federal Regulations
CE
Conformité Européenne
CT Scan
Computed Axial Tomography Scan 
DM
Device Malfunction
EC
Ethics Committee
eCRF
Electronic Case Report Form
EDC
Electronic Data Capture
ESUS
Embolic Stroke of Undetermined Source
FDA
Food and Drug Administration
GCP
Good Clinical Practice
HIPAA
Health Insurance Portability and Accountability Act
HOB
Head of Bed
ICF
Informed Consent Form
IRB
Institutional Review Board
MCA
Middle Cerebral Artery
PFO
Patent Foramen Ovale
RLS
Right to Left Shunt
SAE
Serious Adverse Event
UADE
Unanticipated Adverse Device Effects
TCD
Transcranial Doppler
TEE
Transesophageal Echocardiograph
TTE
Transthoracic Echocardiograph
TIC
Thermal Index for Cranial Bone
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Protocol ID: NA-07BBL-01
Version Date: August 2, 2021
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CONFIDENTIAL
Page 14 of 44
BACKGROUND AND RATIONALE OF STUDY
Background Information
Right to Left shunt (RLS) through a Patent Foramen Ovale (PFO) is a known risk factor of ischemic 
stroke and is present in approximately 25% of the entire population. RLS is responsible for 43% 
of cryptogenic stroke in patients with a mean age of 54 years with no other risk factors2. 
The American College of Cardiology recognizes the benefit of procedures to close PFO in patients 
with PFO-associated stroke for secondary stroke prevention3. With the connection between PFO 
closure and prevention of secondary stroke, there is a need for assessment tools that are both 
non-invasive and accurate. 
Current Options
In the assessment of a possible RLS, the European Society of Cardiology, the European Stroke 
Organization and the European Academy of Neurology recommend either transthoracic 
echocardiogram (TTE) or transcranial Doppler (TCD) imaging of patients with cryptogenic stroke 
or embolic stroke of undetermined source (ESUS)4 that represents an updated classification of 
cryptogenic cerebral ischemia including a systematic and thorough diagnostic work-up. All of these 
diagnostic procedures involve the injection of agitated saline with the patient’s performance of a 
Valsalva Maneuver (VM). While TTE and TCD are initial recommended imaging, the PFO 
presence and size should be subsequently assessed using the more invasive transesophageal 
echocardiography (TEE) methodology5. TTE and TEE are ultrasonic imaging methods used to 
evaluate the structure of the heart. TTE is performed non-invasively through the chest wall using 
a handheld transducer. TEE is slightly more invasive and performed under mild sedation, involving 
the guidance of an ultrasound probe down the esophagus for optimal imaging of the heart. Intra-
cardiac echocardiography (ICE) is perhaps the most invasive imaging method and involves the 
insertion of a catheter to image the heart directly.
  
Outside of these structural imaging modalities, TCD involves the monitoring of blood flow in the 
brain; the presence of microbubbles on a TCD exam at rest and/or after VM could indicate the 
presence of RLS. In this way, TCD is able to mimic the occurrence of an ESUS in a diagnostic 
environment. TCD, like TTE, is non-invasive and involves probe placement and manipulation by 
an expert technician.
 
Limitations
Although TTE is the most frequently used non-invasive technique for the assessment of RLS, it is 
limited by its dependence on the expertise of the technician performing the exam. Furthermore, 
its moderate sensitivity (45.1%) but high specificity (99.6%) in RLS indication should not be relied 
upon in the determination of treatment for ESUS patients6. 
While TEE is considered the gold standard for visualization of the PFO after its presence has been 
indicated, its invasive nature can make it difficult for the patient to comply with the necessary steps 
for the VM. Although TEE is contraindicated in patients with dysphagia or esophageal issues, its 
sensitivity and specificity are 95% and 95% for indication of RLS7. 
Like TTE, the diagnostic value of TCD is limited by the expertise of the technician performing the 
exam and the presence of optimal acoustic windows. Signal acquisition is a well-documented 
obstacle in TCD evaluation regardless of pathology and is no different in the case of RLS 
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assessment8. However, unlike TTE, TCD has shown excellent sensitivity and specificity in RLS
detection (96.1% and 92.4%, respectively) against the gold standard of TEE6. 
Rationale
The non-invasive nature and comparable performance to TEE make TCD a viable but under-
utilized screening tool in the assessment of RLS. There exists a need to decrease its user-
dependence in order to allow for its expanded application.
  
Despite TTE’s superior specificity, the high sensitivity of TCD indicates its obvious diagnostic value 
in the detection of RLS6. 
Potential Risks and Benefits
TCD ultrasonography is a safe, non-invasive, and reproducible technique that allows the 
assessment of cerebral blood flow (CBF) in a person. TCD data collection relies on the level of 
experience of a TCD technologist to acquire an accurate and high-quality signal from the 
intracranial vessel(s) of interest, thus per user, the data quality is variable9. NovaSignal has 
developed the NeuralBot Investigational System (NB-IS) to assist in the TCD data collection such 
that consistent high-quality data may be obtained independent of the experience of the user. 
NovaSignal will collect safety and technical feasibility data regarding the use of TCD ultrasound 
via the NeuralBot Investigational System (NB-IS) device in humans. Data collected during this 
study is for research purposes only. No treatment or healthcare decisions will be made based on 
study measurements collected in this study.
DEVICE DESCRIPTION
Lucid M1 Transcranial Doppler System
The Lucid M1 System is an adjunctive, portable, non-invasive, non-ionizing radiation, point-of-
care TCD diagnostic ultrasound system. It is designed to non-invasively measure and display 
CBFV over the head and neck with a reusable, non-sterile 2-MHz hand-held probe. It can also 
be used bilaterally to monitor the blood flow velocity of the vessels insonated via the temporal 
window of the head with a headset with two reusable, non-sterile 2-MHz monitoring transducers. 
The system can also provide an emboli count for emboli detection.
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NeuralBot Investigational System (NB-IS) 
The NeuralBot Investigational System (NB-IS) is comprised of both the Lucid M1 System and the 
NeuralBot accessory. The NeuralBot accessory must be used with the Lucid M1 System and 
cannot operate independently. The NB-IS is a modification of a 510k cleared device called 
NeuralBot Guided Headmount Accessory (K180455;22May2018). The NB-IS is the investigational 
study device used in this study.  
The NB-IS moves two ultrasound probes around the two temporal regions (Right and Left) of the 
head to find the transtemporal window and then optimizes CBFV measurements. The system uses 
TCD data to systematically specify and evaluate probe positions. The NB-IS consists of a head-
support structure that houses two probe positioning modules, a robotic controller unit and 
computer tablets. The NB-IS is non-invasive and does not deliver energy into a subject.
In this study, the NB-IS system will consist of study specific modifications to aid in study workflow. 
These modifications are not FDA cleared; however, the modifications do not impact the data 
collected in the study. This system will be referred to as NB-IS. The modifications do not introduce 
new risks or alter existing risks. All modifications will be tested to specifications per NovaSignal’s 
Design Control standard operating procedures.
     
Intended use of the device
The NovaSignal Lucid M1 System is a medical ultrasound system intended for use as an adjunct 
to the standard clinical practices for measuring and displaying cerebral blood flow velocity within 
the major conducting arteries and veins of the head and neck. Additionally, the Lucid M1 System 
measures the occurrence of transient emboli signals within the blood stream.
The Lucid M1 System is an FDA cleared diagnostic medical device, Lucid M1 Transcranial 
Doppler Ultrasound System (510k K160442; CE Mark 32518: 21Oct2016). 
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The NeuralBot Investigational System when used with the Lucid M1 System is a medical 
ultrasound device which assists the user in the setup and acquisition of cerebral blood flow velocity 
via the patient’s temporal windows. It is intended for use as an adjunct to standard clinical 
practices for measuring and displaying cerebral blood flow velocity and the occurrence of transient 
emboli within the blood stream.  
The NeuralBot Investigational System is a modification of a 510k cleared device called NeuralBot 
Guided Headmount Accessory (K180455; 22May2018). The NeuralBot Investigational System is 
the 
investigational 
study 
device 
used 
in 
this 
study.
  
The NeuralBot Investigational System is intended to be used by persons qualified by training in its 
safe and effective use. The device is not intended to replace other means of evaluating vital 
patient physiological processes, is not intended to be used in fetal applications and is not intended 
to be used inside the sterile ﬁeld.
The NeuralBot Investigational System is intended to be used on study subjects in the supine (0 
degrees) or up to 45-degrees reclined position on a bed or gurney in a clinical setting. The device 
is NOT intended to be used simultaneously with head imaging equipment such as MRI and CT.
The NeuralBot Investigational System is for use in this study only and is not cleared for use by the 
FDA. It is labelled with the following statement:
CAUTION: Investigational device. Limited by Federal (or United States) law to investigational use 
only. 
The use of the Lucid M1 System with the NeuralBot Investigational System in this study is not for 
the purposes of diagnosis. This device is being used solely for research to develop technology for 
assisted TCD signal acquisition. As such, the device is considered a Nonsignificant Risk Device 
under 21 CFR 812.3(m).
Contraindications
The Lucid M1 System is not intended to be used in fetal applications or inside a sterile field. Use 
proposed in this study is consistent with the current FDA cleared and CE Mark approved labeling. 
The NeuralBot Investigational System is not intended to be used when the following conditions 
are present: 
•
The NeuralBot Investigational System is not intended to be used in persons younger than 18 
years of age.
•
The NeuralBot Investigational System is not intended to be used in fetal applications.
•
The NeuralBot Investigational System is not intended to be used inside the sterile field.
Study Device Training Requirements 
In addition to the protocol training, all planned NB-IS TCD operators will be required to undergo 
training via a device in-service, which includes detailed reviews of the Lucid M1 System and NB-
IS User Manuals and the study device as well as hands-on practice scanning with volunteer 
subjects.
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NovaSignal will provide training for the use of the NeuralBot Investigational System, in adherence 
to the Instructions for Use/User Manual. Documentation of training should be maintained by the 
Principal Investigator throughout the study. Principal investigators will also develop clinical 
guidance training modules so as to standardize the bubble study protocol, including ensuring and 
documenting adequate Valsalva.
Device Storage and Accountability
The NeuralBot Investigational System specific serial numbers must be documented at a study site 
by a designated person, handled and stored properly in a secured location in which only the study 
staff have access to. The Principal Investigator must maintain an accurate record of the status of 
the product(s) throughout the study. Investigators are responsible for appropriate logging of the 
devices used, verification of packing slip information (i.e. lot numbers and quantity shipped), date 
and identity that each device was used in the study, disposition information regarding disposal or 
return to the Sponsor.
STUDY OBJECTIVES
To evaluate the shunt detection rate of the NeuralBot Investigational System (NB-IS) TCD relative 
to 
standard 
of 
care 
diagnostic 
techniques 
(transthoracic 
echocardiography 
(TTE), 
transesophageal echocardiography (TEE), and manual transcranial Doppler ultrasound (TCD)) 
and to assess the safety, accuracy, and usability of the NB-IS device.
Primary Efficacy Endpoint
•
% detection of RLS/PFO with NB-IS TCD compared against standard of care Transthoracic 
Echocardiography (TTE)
Secondary Technical Efficacy Endpoints: 
•
% agreement for detection of RLS/PFO with NB-IS TCD vs SOC TCD
•
% agreement for detection of RLS/PFO with NB-IS TCD vs Transesophageal
Echocardiography (TEE)
•
% agreement for detection of clinically significant shunts with NB-IS TCD vs TTE
•
No window rate (including both unilateral and bilateral absent acoustic windows)
•
Success rate of the NB-IS TCD
•
Incidence of NB-IS device malfunctions
Exploratory Endpoints:
•
Development of an automated algorithm for Spencer Logarithmic Scale10 and International 
Consensus Criteria grading of RLS11
Primary Safety Endpoint:
Incidence of device-related serious adverse events.
Secondary Safety Endpoints:
Incidence of procedure-related adverse events related to agitated saline injection before and after 
VM (headache, allergic reactions, new onset neurological deficit, ischemic stroke, TIA or 
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pulmonary embolism complicating agitated saline contrast injection, etc) will be closely monitored 
and prospectively collected12. 
STUDY DESIGN
This study is a multi-center, prospective, single-arm, non-significant risk (NSR) device study in 
which up to 150 evaluable subjects with embolic stroke of undetermined source (ESUS) according 
to standardized criteria13 will be evaluated with NB-IS TCD and standard of care TTE to screen 
for right to left shunt (RLS) or patent foramen ovale (PFO). Additionally, up to 150 evaluable 
subjects will be evaluated  with NB-IS TCD and standard of care TTE or TEE.
Study Population Size
To ensure the study is adequately powered, up to 150 subjects will be enrolled at up to 7 centers 
in the US. The estimation of this sample size has taken into account the prevalence of suboptimal 
transtemporal windows and potential dropouts.
Once the 150 TTE subjects are enrolled, the study will continue to enroll up to another 150 subjects 
that received a TTE or TEE until enough TTE subjects have been enrolled in the different grades 
of the International Consensus Criteria (0,1,2,3) across the TTE Main Study and TTE/TEE Sub 
Study combined.  
Study Duration
The enrollment period will last up to 15 months. Subject’s participation in the study will last from 1 
to 60 days. 
The study will be complete when all subjects have been enrolled and all data collected. The study 
can be terminated at any time, for any reason, by NovaSignal. Should this occur, the study 
investigator will be notified as soon as possible. The Principal Investigators will be responsible for 
informing their IRBs of the termination of the trial. 
Study Entry Criteria – TTE Main Study
5.3.1
Inclusion Criteria
A subject must meet all of the following inclusions criteria to be enrolled in the study:
1. Subject 18 years of age and older. 
2. Subject presents with a clinical condition characterized by neurological signs and 
symptoms that, in the opinion of the investigator, include embolic stroke or TIA in the 
differential diagnosis.
3. Scheduled for a transthoracic echocardiograph (TTE) study with agitated saline contrast 
(bubble study) within ±30 days of informed consent.
4. Subject is able to successfully perform a Valsalva Maneuver (VM). 
5. Subject or Legally Authorized Representative has the ability to provide informed consent 
and comply with the protocol.  
5.3.2
Exclusion Criteria
A subject cannot be enrolled in the study if any of the following exclusion criteria are met:
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1. Subject has undergone an RLS/PFO closure. 
2. Female who is pregnant or lactating at time of admission
3. Subjects who underwent partial or full craniotomy/craniectomy within the past 6 months. 
4.
Subjects who have a physical limitation preventing TCD/Headmount placement.
Study Entry Criteria – TTE or TEE Sub-Study (after first 150 subjects with TTE enrolled)
5.4.1
Inclusion Criteria
A subject must meet all of the following inclusions criteria to be enrolled in the study:
1. Subject 18 years of age and older.
2. Subject presents with a clinical condition characterized by neurological signs and 
symptoms that, in the opinion of the investigator, include embolic stroke or TIA in the 
differential diagnosis.
3. Scheduled 
for 
a 
transthoracic 
echocardiograph 
(TTE) 
or 
transesophageal 
echocardiograph (TEE) study with agitated saline contrast (bubble study) within ±30 days 
of informed consent.
4. Subject is able to successfully perform a Valsalva Maneuver (VM).
5. Subject or Legally Authorized Representative has the ability to provide informed consent 
and comply with the protocol.  
5.4.2
Exclusion Criteria
A subject cannot be enrolled in the study if any of the following exclusion criteria are met:
1. Subject has undergone an RLS/PFO closure.
2. Female who is pregnant or lactating at time of admission
3. Subjects who underwent partial or full craniotomy/craniectomy within the past 6 months. 
4.
Subjects who have a physical limitation preventing TCD/Headmount placement.
STUDY PROCEDURES
Informed Consent and Screening
Subject identification and eligibility will be determined by the PI or study team based on their 
clinical assessment of the potential subject while that patient is undergoing standard of care for 
their condition. 
Before participation in the study, candidates who may be eligible for the study will be provided 
with an informed consent according to 21 CFR 56 and 45 CFR 160-164 and guidelines of the 
Investigational Review Board of the institution at which the study is being conducted.  Candidates 
will be given time to review the consent form and ask questions about the study. The investigator
or delegated study staff is responsible for obtaining written informed consent from each potential 
study participant. Informed consent should be obtained, when required, in written format and using 
a form approved by the local IRB. 
The subject must receive a copy of the signed and dated informed consent if requested. Waivers 
of consent will not be utilized in this study. The candidate or legal representative must sign the 
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consent form prior to enrollment. Candidates will also be required to sign a Health Insurance 
Portability and Accountability Act (HIPAA) form which includes a Privacy Rule. This rule gives 
special safeguards to Protected Health Information (PHI) that is identifiable or can be directly 
linked to the subject (e.g., social security number, name, birth date). This authorization may be 
part of the informed consent form or separate. If applicable to the investigator site location, 
candidates will also be required to sign the California Experimental Subject Bill of Rights. 
A subject will be considered enrolled at the time of Informed Consent signature.  
Numbering of Study Subjects
Each site will be assigned a site number at the beginning of the study and each enrolled subject 
will be assigned a subject number. The subject number will consist of the study number, the site 
number followed by a sequential number that begins with “001”.
Screen Failures
Subject eligibility will be determined by the PI or study team based on their clinical assessment of 
the potential subject while the patient is undergoing standard of care assessments for a stroke in 
the hospital. 
For this protocol, a single measurement is defined as a collection of data at a single depth on a 
single side of the head. A scan is defined as a set of measurements which occur during a single 
data collection session and can include multiple measurements. A bilateral scan refers to 
measurements which are collected on each side of the head.  
Subjects will be measured with NB-IS TCD and the de-identified data will be reviewed to determine 
the quality of the data for analysis. Subjects are considered evaluable if their NB-IS TCD data is 
of adequate quality and a complete data set is acquired. An adequate, evaluable, and complete
study is defined for the purposes of this protocol as successful acquisition of a unilateral signal at 
40-65mm depth range recorded for the duration of the bubble study in the Resting position and 
with Valsalva Maneuver. If the scan quality is adequate, the subject study data (via eCRF) and 
imaging will be collected and the NB-IS TCD utilized for analysis of the study endpoints. If the
scan quality is inadequate, all subject clinical and imaging data will not be included in the final 
analysis.
Study Discontinuation by IRB or Sponsor
The IRB may choose to discontinue the study at any center(s) for which they granted approval if 
the: 
•
The research study is not conducted in accordance with the IRB requirements. 
•
The research study indicates unexpected serious harm to Subjects. 
The Sponsor may choose to discontinue the study should the Sponsor discover additional 
information during the study that may cause harm to subject safety. 
If the study is terminated prematurely or suspended, the Sponsor will promptly inform all clinical 
Investigators of the termination or suspension and the reason(s) for this. The IRB/EC will also be 
informed, either by the Sponsor or Investigator if a local IRB/EC is utilized, promptly and provided 
with the reasons(s) for the termination. If applicable, regulatory authorities will be informed.
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Study Schedule
6.5.1
Schedule of Assessments
Schedule of Assessments
Assessments
Screening1
Procedure1
Follow-Up1
Informed Consent
X 
Demographics
X 
Relevant Medical History
X 
Physical Exam ♦
X
SOC TCD Bubble Study Exam ♦
O4 
NB-IS TCD Bubble Study Exam
X2
Transthoracic Echocardiograph (TTE) Bubble Study ♦
X3 
Transesophageal Echocardiograph (TEE) ♦
O4 / X8
PFO Closure Decision ♦
X5
Classification of Potential Causative Mechanism in 
PFO–Associated Stroke7♦
X 
PFO Closure Procedure ♦
O6
Device-Related Adverse Events
X 
Procedure-Related Adverse Events
X 
Concomitant Medications
X 
X 
Device Deficiencies
X 
♦Standard of Care
X = Required
O = Optional, if performed
1.
Screening, Procedure, and Follow-Up Visits can occur on the same day
2.
NB-IS TCD performed with contrast agent of 9 ml bacteriostatic saline with 1 ml air mixed 10 times. Performed 
at Rest AND with VM – 60 sec recordings each in supine positions AND with HOB raised to 45 degrees.
3.
TTE can be performed within ±30 days of informed consent per SOC. 
4.
SOC TCD and TEE results collected if performed as applicable per standard of care.
5.
PFO closure decision collected post diagnostic imaging procedures.
6.
PFO closure procedure data collected within 60 days post diagnostic workup, if performed
7.
Elgendy AY, Saver JL, Amin Z, et al; MSCAI14. Proposal for Updated Nomenclature and Classification of 
Potential Causative Mechanism in Patent Foramen Ovale-Associated Stroke. JAMA Neurol. 2020 Apr 13. 
doi:10.1001/jamaneurol.2020.0458. [Epub ahead of print] PubMed PMID: 32282016.
8.
SOC TTE or TEE will be required after the first 150 TTE subjects are enrolled and can be performed within ±30 
days of informed consent per SOC.
Scanning/Data Acquisition  
The User Manual provided for the device details, in specific steps, the actions to be executed 
with the device for scanning/data acquisition. A general overview is provided below. 
NeuralBot Investigational System (NB-IS) Scanning Technique
The NB-IS TCD scanning session will consist of an initial set-up and signal search (up to 20 
minutes). During this time, the system will search for CBFV signals at depths between 40-65 mm. 
Once the signal is acquired, the system can monitor the signal for up to 1 hour. In this study, once 
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the MCA signal has been acquired unilaterally or bilaterally, subjects will be monitored for up to 
20 minutes during the delivery of the contrast agent at Rest and with Valsalva Maneuver. 
The head cradle of the NeuralBot Investigational System is positioned underneath the subject’s 
head and secured to a fixed location (bed, chair, or gurney). The user will affix two registration 
dots on each side of subject’s temples. The subject’s head is then positioned into the head cradle. 
The TCD modules are aligned to the head such that the probes are in contact with the subject’s 
temples (temporal window). The user will register the Headmount to the subject’s specific head 
shape and size. The user will apply gel to improve signal quality. The NeuralBot Investigational
System will collect data and indicate to the user the scan status. 
Subjects will be evaluated during the scanning period per ALARA (“As Low As Reasonably 
Achievable”) criteria. Scanning may not violate ALARA levels and will be immediately terminated 
if ALARA criteria reached during a scanning session. 
ALARA Considerations
ALARA Considerations: “As Low As Reasonably Achievable – ALARA” principle for ultrasound to 
reduce the amount of total exposure to the subject without compromising exam quality.
Diagnostic ultrasound, including TCD, has been used clinically and in research for decades. In 
addition to the FDA guidance, there are several groups including the American Institute of 
Ultrasound in Medicine (AIUM), British Medical Ultrasound Society (BMUS), and the World 
Federation for Ultrasound in Medicine and Biology (WFUMB) that make recommendations on 
ultrasound safety for both fetal and non-fetal applications. These groups have focused on several 
areas of ultrasound safety including Thermal Index (TIC). 
The FDA specifically references the AIUM for the ALARA recommendations for TIC and 
corresponding scan durations in the new guidance released in October 2017. Table 1 shows the 
recommended scan duration for a given TIC range. The ALARA recommendations are based on 
the TIC output by the device (which is displayed on the Lucid M1 System device at all times). TIC 
is defined as:
TIC – the thermal index for applications in which the ultrasound beam passes through bone near 
beam entrance into the body. 
Thermal Index (TIC)
Recommended Duration
(minutes)
>6.0
0
5.0-6.0
0.25
4.0-5.0
<1
3.0-4.0
<4
2.5-3.0
<15
2.0-2.5
<60
1.5-2.0
<120
<1.5
No Limit
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Table 1 - Recommended maximum exposure duration and TIC ranges (except the eyes which are 
not evaluated in this protocol)
If a subject meets ALARA criteria at any time during a scanning session, the scan will be 
discontinued.
Standard of Care Procedures
Most procedures completed as part of this study are considered standard practice and include 
relevant medical and medication history, physical exam, SOC transcranial Doppler ultrasound 
(TCD) bubble study, transthoracic echocardiography (TTE) bubble study, transesophageal 
echocardiography (TEE) bubble study, and PFO closure (status), if performed.  
This study is incorporating the use of NB-IS TCD bubble study in conjunction with standard of care 
procedures.
Study-Specific Procedures
The procedures that are study-specific and not part of standard of care for diagnosis and treatment 
of RLS and PFO include the use of the NB-IS TCD System bubble study. 
Screening Procedures
The following data will be collected, and assessments performed after obtaining informed consent 
from eligible subjects and prior to the NB-IS TCD procedure.
•
Informed Consent
•
Subject Demographics
•
Relevant Medical History
•
Physical Exam (SOC)
•
Relevant Concomitant Medications
Procedure –NB-IS TCD Scanning Session
The NB-IS TCD bubble study can be performed during the same session as SOC TCD bubble 
study is performed and preferably before TTE and TEE (if applicable) bubble studies are 
performed. If a TTE bubble study is performed before TCD bubble study, the SOC TCD operator 
will ensure he/she are blinded to the results of the TTE prior to performing TCD. The NB-IS TCD 
operator will perform scanning sessions as follows:
6.12.1 Patient Preparation – Valsalva Maneuver (VM)
The study subject will be trained in performance of the VM prior to conducting the SOC TCD (if 
performed) and NB-IS TCD bubble study in accordance with the training module developed by the 
principal investigators. 
6.12.2 Contrast Agent Preparation
For TCD embolus detection, the contrast agent consisting of 9 mL bacteriostatic saline solution 
and 1 mL air should be mixed between two (2) 10-mL syringes connected by a 3-way stopcock at 
least ten (10) times and then vertically injected into (ideally) the right antecubital vein as a bolus 
both at Rest and mid-way through injection before a ten second Valsalva Maneuver (VM). The 
agitated saline should be mixed with 1 mL of blood prior to injection, if possible14,15,16. If Definity
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(ultrasound contrast-agent containing perflutren lipid microspheres) is given during TTE or TEE 
prior to any TCD (NB-IS or SOC) must use different IV, wait up to 24h from Definity injection and 
record without injection (baseline recording) for 5 minutes prior to injecting air contrast (screen for 
Definity still circulating) before SOC TCD and NB-IS TCD bubble study sessions. 
6.12.3 SOC Transcranial Doppler Ultrasound Procedure (TCD) (as applicable) 
If SOC TCD bubble study is performed during subject participation in the study, preference is for 
this exam to be conducted in same session as the NB-IS TCD with the same individual 
administering the bubble injections with the SOC TCD bubble exam occurring before NB-IS TCD. 
The SOC TCD bubble study will be performed per standard of care, however, if possible, 
preference would be to collect measurements similar to NB-IS TCD exam (e.g., supine position at 
Rest and with VM, 45 degrees at Rest and with VM, etc.). 
 
The following data points will be collected for the SOC TCD procedure: 
 
• 
SOC transcranial Doppler Ultrasound (TCD) bubble study local results (SOC) 
o Date/time of SOC TCD bubble study 
o SOC TCD scan Start/Stop times with and without VM (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
o Head inclination (degree)/subject positioning/location of the exam 
o Local Positive/Negative results for RLS/PFO 
o Local Spencer Logarithmic Scale (SLS) grade (with and without VM) 
o Local International Consensus Criteria (ICC) grade (with and without VM) 
• 
 
6.12.4 NeuralBot Investigational System (NB-IS) TCD Procedure 
The NB-IS TCD bubble study session will be conducted beginning in the supine position with the 
same IV line. 
 
The NB-IS TCD operator will setup the study subject in the headmount of the NeuralBot 
Investigational System as per Section 6.7 NeuralBot Investigational System Scanning Technique. 
When the signal search has completed and the optimal signal has been identified unilaterally or 
bilaterally , the NB-IS device will continue monitoring the signal(s) for up to 20 minutes during the 
delivery of the contrast agent at Rest and with VM. 
 
First, 10 mL of mixed contrast agent will be injected into the right antecubital vein while the subject 
is at rest and the MCA signal recorded for a period of 60 seconds. The subject will remain at rest 
for approximately 3 to 5 minutes or when bubbles have cleared. 
 
Second, another 10 mL of contrast agent will be injected in the right antecubital vein and the VM 
performed mid-way through injection, sustained for ten seconds. The MCA signal will be recorded 
for a period of 60 seconds; characteristic MCA waveform morphology change and mean velocity 
decrease of at least 25% will serve as proof of adequate Valsalva effort17. 
 
This procedure will be repeated with the study subject in the reclined position, with head of bed 
raised to 45 degrees where 10 mL contrast agent is injected when the subject is at rest and 
subsequently with a Valsalva Maneuver.  
 
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The following data points will be collected for the NB-IS TCD scanning session: 
 
• 
Date/Time of NB-IS TCD bubble study 
• 
NB-IS TCD Operator (name) 
• 
NB-IS TCD Scan information 
o NB-IS TCD scan Start/Stop times with and without VM 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM 
o Head inclination (degree)/subject positioning/location of the exam 
• 
Relevant Concomitant Medications 
• 
Device-Related Adverse Events (NB-IS) 
• 
Procedure-related adverse events related to agitated saline injection before and after VM 
(headache, allergic reactions, new onset neurological deficit, ischemic stroke, TIA or 
pulmonary embolism complicating agitated saline contrast injection, etc) will be closely 
monitored and prospectively collected18. 
• 
Device Deficiencies / Malfunctions / Technical Observations 
6.12.5 Transthoracic Echocardiography Procedure (TTE) per SOC 
The TTE bubble study can be performed per standard of care within ±30 days of informed consent. 
 
The following data points will be collected for the TTE procedure: 
 
• 
Transthoracic Echocardiography (TTE) local results (SOC) 
o Date/Time of TTE bubble study 
o TTE Start/stop times with and without VM (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
o Local Positive/Negative results for RLS/PFO 
o Local Bubble Count (as applicable) 
o Local TTE grading (as applicable) 
6.12.6 Transesophageal Echocardiography Procedure (TEE) per SOC (as applicable) 
The study subject, if referred per the institution’s standard practice, will then undergo scheduled 
transesophageal echocardiography (TEE) bubble study with and without VM similarly to NB-IS 
TCD procedure. 
 
The following data points will be collected for the TEE procedure, if performed: 
 
• 
Transesophageal Echocardiography (TEE) local results (SOC) including shunt size and 
presence of atrial septal aneurysm. 
o Date/Time of TEE exam 
o TEE Start/Stop times with and without VM (as applicable) 
o Saline/Air Injection(s) Start/Stop time(s) with and without VM (as applicable) 
o Local Positive/Negative results for RLS/PFO 
o Local Bubble Count (as applicable) 
o Local TEE grading (as applicable) 
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6.12.7 Follow-Up Procedures
After the NB-IS TCD, TTE, SOC TCD and TEE (if performed) bubble study exams performed, the 
following data points will be collected:
•
PFO Closure Decision (post-diagnostic procedure(s))
•
Classification of Potential Causative Mechanism in PFO–Associated Stroke1
If a PFO closure procedure is performed within 60 days post diagnostic workup, the following 
data points will be collected:
•
PFO Closure Procedure Data
•
Shunt size
•
Closure device(s) used
NB-IS TCD data collection and data transfer
NB-IS TCD data will be collected and stored on the NeuralBot Investigational System. The details 
of the data collection and transfer can be found in the device User Manual and Data Transfer 
procedures. 
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Study workflow
Standard of Care
Arrive at Neuro Unit
Pre-Screened for Enrollment
1) Scheduled SOC TTE
2) ≥ 18 years of age
Transesophageal Echocardiograph (TEE)
Optional Main Study / Required Sub-Study
NB-IS TCD
All Clinical Data Points Collected – EMR
NB-IS TCD/SOC TCD/TTE/TEE Data to 
CORE LAB
NB-IS TCD Data to NovaSignal
Subject meet all I/E criteria?
SOC TCD
Yes
No
SOC TCD/ TTE/TEE Results
PFO Closure Decision &
PFO-Associated Stroke Classification
INFORMED CONSENT
Pre-Screen
Failure
Study Procedure
NB-IS TCD Procedures:
1. Performed with contrast agent of 9 
ml saline with 1 ml air  mixed 10 
times
2. Performed at Rest AND with VM – 
60 sec recordings each in supine 
positions AND with HOB at 45 
degrees.
Transthoracic Echocardiograph (TTE)
PFO Closure Procedure, if performed
Study Procedure
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RISKS AND BENEFITS
There is minimal potential clinical risk associated with the scans described in the study. The risks 
associated with the use of the NeuralBot Investigational System in humans have not been 
determined as this study is one of the initial uses of the device. An ongoing study utilizing the 
device in subjects experiencing neurological symptoms of stroke has not reported any unexpected 
or serious device related adverse events (Protocol No.: NA-01STR-01). The NeuralBot 
Investigational System is designed to be physically and electro-mechanically safe according to 
manufacturing standards. At any time, for any reason, the use of the device can be discontinued.
Known Potential Risks
The following device-related adverse events have been identified as possible (anticipated) with 
the use of the NeuralBot Investigational System: 
•
Physical discomfort of the head and neck due to pressure from the probe, or probe 
accidentally coming in contact with ears, eyes or hair. 
•
Psychological feeling of claustrophobia- the fear of being enclosed in a small space. 
Benefits
There is no direct benefit to the subject participating in the study. 
SAFETY ASSESSMENTS
Safety Parameters
8.1.1
Adverse Event (AE)
An adverse event (AE) is defined as any untoward medical occurrence, unintended disease or 
injury, or untoward clinical signs (including abnormal laboratory findings) in subjects, users, or 
other person, whether or not related to the investigational medical device (ISO14155:2011E).
•
Note 1: This definition includes events related to the investigational medical device or the 
comparator
•
Note 2: This definition includes events related to the procedures involved
•
Note 3: For users or other persons, this definition is restricted to events related to the study
device.
For the purpose of this protocol, adverse events will be reported and recorded (via eCRF) 
if any of the following apply:
•
Causality is related to:
o
the study device
o
the study-related procedure (not standard of care)
o
if causality is unknown
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• 
The event is a serious adverse event (SAE) related to the study device or study-related 
procedure 
 
Any medical condition that is present at the time informed consent is obtained or prior to the start 
of the study procedure will be considered as baseline and not reported as an AE. Such conditions 
should be added to the medical history, if not previously reported. 
 
Collection of device and/or procedure-related adverse events will start after the time that informed 
consent form is obtained. Device and procedure-related adverse events will be monitored 
throughout the study. 
8.1.2 Serious Adverse Event (SAE) 
A serious adverse event (SAE) is defined (ISO14155:2011E) as an adverse event that: 
 
a) Led to a death 
b) Led to a serious deterioration in the health of the subject that: 
1) resulted in a life-threatening illness or injury, or 
2) resulted in a permanent impairment of a body structure or a body function, or 
3) required in-patient or prolongation of hospitalization, or 
4) resulted in medical or surgical intervention to prevent life-threatening illness or 
injury or permanent impairment to body structure or a body function 
c) Led to fetal distress, fetal death or a congenital abnormality or birth defect. 
 
NOTE: Planned hospitalization for a pre-existing condition, or a procedure required by the 
protocol, without serious deterioration in health, is not considered a serious adverse event.  
8.1.3 Unanticipated Adverse Device Effect (UADE/USADE) 
An Unanticipated ADE (UADE) is any serious adverse effect on health or safety or any life-
threatening problem or death caused by or associated with a device, if that effect, problem, or 
death was not previously identified in nature, severity, or degree of incidence in the protocol or 
application (including a supplementary plan or application (e.g., user manual, investigator’s 
brochure, instructions for use, etc.)); or any other unanticipated serious problem associated with 
a device that relates to the rights, safety, or welfare of subjects (ISO14155:2011E). 
8.1.4 Device Deficiency, Device Malfunction, and Use Error 
Device Deficiency 
Defined as inadequacy of a medical device with respect to its identity, quality, durability, reliability, 
safety, or performance 
Device Malfunction 
Defined as a failure of a medical device to perform in accordance with its intended purpose when 
used in accordance with the instructions for use or protocol or user manual. 
Use error 
Defined as the act or omission of an act that results in a different medical device response than 
intended by the manufacturer or expected by the user. 
 
• 
Note 1: Use error includes slips, lapses, and mistakes. 
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•
Note 2: An unexpected physiological response of the subject does not in itself constitute a 
use error.
Device deficiencies are also reportable if they did not lead to an adverse event but could have led 
to a SADE:
a) if either suitable action had not been taken,
b) if intervention had not been made, or
c) if circumstances had been less fortunate
(ISO14155:2011E)
Reporting Device and/or Procedure Related Adverse Events
All suspected device related adverse events shall be recorded on the Adverse Event page of the 
CRF if they are suspected to be related to the use of the NeuralBot Investigational System. If 
appropriate, the event shall subsequently be reported to the relevant IRB. The event shall be 
thoroughly investigated and a causal relation as to whether the event is related or not to the use 
of the NeuralBot Investigational System shall be established.
The Investigator will record the nature, severity, relatedness, treatment, and outcome of the AE. 
This classification of the event determines the reporting procedures to be followed. NovaSignal
may upgrade the classification as required for reporting purposes.
At the initiation of device use through end of use of the device, all subjects with adequate quality 
NeuralBot Investigational System data shall be assessed for any potentially device related 
complications or adverse events. All events shall be followed until resolution or through the end 
of a subject’s study participation. 
Device-related adverse events information will be collected throughout the study. From the 
initiation of and end of device use, all subjects scanned with the NeuralBot Investigational System 
shall be assessed for any potentially device related complications or adverse events. All events 
shall be followed until resolution or through the end of a subject’s study participation. 
Procedure-related adverse events related to agitated saline injection before and after VM 
(headache, allergic reactions, new onset neurological deficit, ischemic stroke, TIA or pulmonary 
embolism complicating agitated saline contrast injection, etc) will be closely monitored and 
prospectively collected18. Nevertheless, it should be noted that a recent multi-center study 
underlined the safety and importance in performing TCD-BS for RLS detection in a standardized 
manner using the currently recommended international consensus protocol with agitated saline 
intravenous injections19. 
Event, onset date, severity, relatedness, device relationship, treatment and outcome of the AE will 
be recorded on the appropriate case report form. Any device-related and/or procedure-related
AEs will be monitored until they are adequately resolved or explained. This classification of the 
event determines the reporting procedures to be followed. For purposes of this protocol, the 
following definitions will apply.
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Classification of a Device and/or Procedure Related Adverse Event
8.3.1
Severity Rating of Event
The following categories of adverse event severity are to be used:
Mild 
Awareness of sign or symptom that does not interfere with the subject’s usual 
activity or is transient, resolved without treatment and with no sequelae.
Moderate
Interferes, but does not hinder, the subject’s usual activity and/or may require 
treatment.
Severe
Symptom(s) causing severe discomfort and significant impact on the subject’s 
usual activity and requires treatment or intervention.
8.3.2
Relationship of Study Device and/or Procedure
The causal relationship to study device and/or procedure will be evaluated as follows:
Caused By
Relation
Definition of Relation
Study 
Device
Definitely 
Related
The event is clearly related to the study device beyond 
reasonable doubt.
Probably 
Related
The event is temporally associated and plausibly related to the 
study device but there are also potential alternative 
explanations, though the alternatives are not likely.
Possibly 
Related
The adverse event may be related, scientifically plausible to 
the study device, but there are also alternative explanations.
Procedure
Definitely 
Related
The event is clearly related to the procedure beyond 
reasonable doubt.
Probably 
Related
The event is temporally associated and plausibly related to the 
procedure but there are also potential alternative explanations, 
though the alternatives are not likely.
Possibly 
Related
The adverse event may be related, scientifically plausible to 
the procedure, but there are also alternative explanations.
8.3.3
Outcome of Event
The outcome of each Device and/or Procedure Related Adverse Event must be assessed 
according to the following classifications:
Classification
Definition
Resolved
Subject fully recovered with no observable residual effects
Resolving
Subject’s condition improved, but residual effects remain
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Resolved with Sequelae
Subject recovered with observable residual effects
Ongoing
Event is ongoing without changes in the overall condition
Fatal
Subject died as a result of the device and/or procedure related 
event
Reporting Procedures
All UADE/SADE/SAEs must be recorded and reported to the Sponsor via the eCRFs throughout 
the study, immediately upon study site staff awareness of the event but not later than 72 
hours by the study site personnel. In the event the EDC is unavailable, events can be reported 
via email to the Sponsor study mailbox: events@novasignal.com and/or to the study Sponsor 
contact. Note: The event will still need to be recorded on the eCRFs once the EDC is functional. 
In case device malfunctions occur, they will be reported to NovaSignal within 10 business days. 
The report should include at a minimum, a description of event, date of occurrence, lot or serial 
number of the device. 
Timing for the reporting for the different types of AEs and Device Deficiencies is described as 
follows:
Classification
Reporting time
Type of report
Unanticipated Adverse 
Device Effects (UADE)
Notify NovaSignal immediately upon 
study site staff awareness of event but 
not later than 72 hours. Notify IRB as 
required. 
Device/Procedure
Related Serious 
Adverse Event Report 
Form
Serious Adverse Events 
(SAE) or Serious 
Adverse Device Effect 
(SADE)
Notify NovaSignal immediately upon 
study site staff awareness of event but 
not later than 72 hours. Notify IRB as 
required.
Device/Procedure
Related Serious 
Adverse Event Report 
Form
Study Device 
Deficiency/Malfunction
With or Without AE
Notify NovaSignal within 10 business 
days of learning of event. Notify IRB as 
required.  
Device Deficiency/
Malfunction Form
PROTOCOL DEVIATIONS
A protocol deviation is defined as a failure to comply (intentionally or unintentionally) with the 
requirements of the clinical study as specified in the protocol.
Examples of deviations include but are not limited to a required test not being done or not being 
done within the specified timeframe, a subject enrolled who did not meet the inclusion/exclusion 
criteria, or enrolment of a subject without appropriate consent. 
  
Deviations from the protocol must be reported to NovaSignal through the eCRFs and will be 
reviewed and assessed by the Sponsor.
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The site is responsible to use vigilance to identify and report deviations to the Sponsor and per 
site IRB policies. The study monitors shall verify that the conduct of the study is in compliance 
with the approved protocol and applicable regulations and shall identify deviations and any issues 
of noncompliance. Corrective and preventative actions will be implemented promptly and as 
necessary. Significant protocol deviations that raise subject safety concerns or indicate repeat 
noncompliance may be grounds for investigator disqualification.
The Investigator must provide reports of the progress, completion, termination or discontinuation 
of the study to the IRB(s) at appropriate intervals as designated by the Sponsor and per IRB 
requirements.
QUALITY CONTROL AND QUALITY ASSURANCE 
Organizational Preparations 
A site evaluation via personnel and facility documentation will be performed by NovaSignal or their 
designee during the study to ensure the availability of appropriately trained personnel to conduct 
the study according to the FDA Code of Federal Regulations and ICH Guidelines on Good Clinical 
Practices (ICH-E6). This study will be conducted under the principles described in the Declaration 
of Helsinki. 
Training 
NovaSignal will provide training for the use of the NeuralBot Investigational System, in adherence 
to the Instructions for Use/User Manual. Documentation of training should be maintained by the 
Principal Investigator throughout the study. 
Data Quality Assurance
Training of appropriate site personnel will be the responsibility of NovaSignal or designee. To 
ensure uniform data collection and protocol compliance, site personnel will utilize source 
documentation worksheets to document protocol procedures. Principal investigators will assist in 
developing training modules for standardization of clinical technique.
Subject Privacy 
All data will be maintained under highly secure and fully HIPAA-compliant dedicated servers under 
the direction supervision of the Principal Investigator. Primary data that is obtained will have 
identifying information and will be stored securely onsite. Access to identifiable subject data is 
limited to project staff who have direct data management and/or statistical responsibilities, under 
the direct supervision of the Principal Investigator.
The study participant’s contact information will be securely stored at each clinical site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by the reviewing IRB.
Data de-identification will be performed manually using the "safe-harbor" approach under the 
supervision of the Principal Investigator. At this point, a unique ID is generated for each subject, 
and each exam. These will populate two databases using the "safe-harbor" approach under the 
supervision of the Principal Investigator. The database that is devoid of all PHI will be used for 
data analysis in this research.
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Case Report Forms
All required data for this study will be collected on standardized Case Report Forms (CRF). The 
forms may be paper based or electronic. The forms will only include the subject study number 
assigned once the subject is screened. The investigator is responsible for the accuracy, 
completeness and legibility of the data reported to NovaSignal in the CRFs and in all required 
reports. The CRFs are to be dated and signed by an investigator on appropriate pages to verify 
that he/she has reviewed the recorded data. 
Investigational Device Accountability  
The NeuralBot Investigational System specific serial numbers must be documented at a study site 
by a designated person, handled and stored properly in a secured location in which only the study 
staff have access. The Principal Investigator must maintain an accurate record of the status of the 
products throughout the study. Investigators are responsible for appropriate logging of the devices 
used, verification of packing slip information (i.e. lot numbers and quantity shipped), date and 
identity that each device was used in the study, disposition information regarding disposal or return 
to the Sponsor.
Selection of Investigators
The study will be conducted at multiple centers. Study staff for each center will meet the following 
criteria:
•
A user trained by NovaSignal in the use of the Lucid M1 System and NeuralBot 
Investigational System.  
•
Commitment from the participating investigator to pursue details of any potentially device
and procedure related adverse event outcomes. 
•
Commitment from the participating investigator to enroll only subjects meeting inclusion 
and exclusion criteria of the local approved protocol. 
• 
Dedicated staff members who can collect and transmit data and be willing to maintain all 
study-related documentation (e.g., source documents, CRFs, regulatory documentation, 
etc.) 
Close-out Document Review
The purpose of the final document review is to collect all outstanding study data documents, 
ensure that the principal investigator’s files are accurate and complete, review record retention 
requirements with the principal investigator and ensure that all applicable requirements are met 
for the study.
The investigator agrees to allow the monitoring of study data, the completion of all data clarification 
or audits even after study close-out visit has been performed at NovaSignal’s request.
STATISTICAL ANALYSIS 
Primary Endpoint Analysis
•
Demonstration of NB-IS TCD diagnostic performance for shunt detection, as compared to 
transthoracic echocardiography (TTE) for diagnostic accuracy.
o
Diagnostic Accuracy - Shunt detection rate (%)
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o
% detection of true positives (TP) (sensitivity), all sample size estimations will be 
computed with regard to the endpoint of sensitivity
Secondary Technical Efficacy Endpoint(s) Analysis
•
% detection of false positives (FP), comparison of NB-IS TCD with TTE
•
% detection of true negatives (TN) (specificity), comparison of NB-IS TCD with TTE
•
% detection of false negatives (FN), comparison of NB-IS TCD with TTE
•
Positive Predictive Value (PPV), comparison of NB-IS TCD with TTE
•
Negative Predictive Value (NPV), comparison of NB-IS TCD with TTE
•
Demonstration of NB-IS TCD diagnostic performance to SOC TCD for diagnostic accuracy
parameters (sensitivity, specificity, PPV, NPV). 
o
If bilateral signals are found, the microbubble count will be sum across both vessels. 
o
If only a unilateral signal is found, the microbubble count will be doubled for the total count.
o
Spencer Logarithmic Scale (SLS) and International Consensus Criteria (ICC) gradings 
from the Imaging Core Lab for NB-IS TCD and SOC TCD will be compared for analysis.
•
Demonstration of NB-IS
TCD diagnostic performance
targeting to transesophageal 
echocardiography (TEE) for diagnostic accuracy parameters (sensitivity, specificity, PPV, 
NPV). 
•
% detection of intervenable shunts, comparison of NB-IS TCD with TTE
o
For NB-IS TCD, Grade 3 and above (>30 microbubbles) on the Spencer Logarithmic Scale 
(SLS) and International Consensus Criteria (ICC) considered intervenable shunt10. 
o
For TTE, >20 microbubbles per 2016 American Society of Echocardiography Guidelines 
and Standards23 considered intervenable shunt. 
•
NB-IS TCD No Window rate performance to SOC TCD parameters (including both unilateral
and bilateral absent acoustic windows).
•
% success rate of NB-IS TCD
o
Incidence of failed registrations (device unable to register)
o
Incidence of bilateral signals not found (no evaluable signals found both sides)
o
Incidence of unilateral signals not found (evaluable signal found on one side)
o
An adequate, evaluable, and complete study is defined for the purposes of this protocol as 
successful acquisition of a unilateral signal at 40-65mm depth range recorded for the 
duration of the bubble study in the Resting position and with Valsalva Maneuver.
•
Incidence of device malfunctions 
Exploratory Endpoints Analysis
•
Demonstration of NB-IS TCD accuracy to Spencer Logarithmic Scale (SLS) Grade and 
International Consensus Criteria (ICC) grade for diagnostic accuracy.
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Imaging Core Lab Analysis
All de-identified imaging data will be sent to a core laboratory which will provide independent 
quantitative and qualitative assessment of all NB-IS TCD and SOC TCD, TTE, and TEE bubble 
study data. They will be blinded to the study and local diagnostic report data and provide 
independent review. The Core Lab interpretations will supersede all local interpretations and will 
be applied to all study endpoint analyses as applicable.
RLS/PFO Validation and Analysis 
Evaluation of correlation between diagnostic imaging standard of care (SOC TCD/TTE/TEE) and 
NB-IS TCD diagnostic performance by an independent core laboratory. Success criteria will 
include clinical validation of NB-IS TCD for RLS/PFO detection targeting (i) 40% increase in NB-
IS TCD sensitivity compared to TTE sensitivity and (ii) NB-IS TCD sensitivity ≥90% compared to 
SOC TCD sensitivity.
Sample Size Calculation
The present study was powered based on the results of a recent meta-analysis reporting a pooled 
TCD sensitivity of 96.1% for RLS detection, while the pooled TTE sensitivity was estimated at 
45.1% (absolute difference of 51%)6. For power calculations, we used a more moderate effect 
size of 40% increase in the sensitivity of NB-IS TCD compared to TTE. 
A sample size of 100 subjects achieves 90% power to detect a difference of 40% between two 
diagnostic tests whose sensitivities are 90% (TCD) and 50% (TTE). This procedure uses a two-
sided McNemar test with a significance level of 0.05. The mean prevalence of PFO in the 
population of patients with cryptogenic stroke is at least 30%2. The proportion of discordant pairs 
has been set at 0.500.
        Sensitivity      Sensitivity Proportion
 
 
of Test 1
of Test 2
Difference Discordant  Prevalence
Power
N 
Se1
Se2
Se1-Se2
D  
P  
Alpha Beta
0.00000
10
0.900
0.500
0.400
0.500
0.300
0.05000
1.00000
0.00830
20
0.900
0.500
0.400
0.500
0.300
0.05000
0.99170
0.12990
30
0.900
0.500
0.400
0.500
0.300
0.05000
0.87010
0.32013
40
0.900
0.500
0.400
0.500
0.300
0.05000
0.67987
0.49003
50
0.900
0.500
0.400
0.500
0.300
0.05000
0.50997
0.62345
60
0.900
0.500
0.400
0.500
0.300
0.05000
0.37655
0.72404
70
0.900
0.500
0.400
0.500
0.300
0.05000
0.27596
0.79937
80
0.900
0.500
0.400
0.500
0.300
0.05000
0.20063
0.85654
90
0.900
0.500
0.400
0.500
0.300
0.05000
0.14346
0.89995
100
0.900
0.500
0.400 
0.500
0.300
0.05000
0.10005
0.93182
110
0.900
0.500
0.400
0.500
0.300
0.05000
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0.06818 
0.95405 
120 
0.900 
0.500 
0.400 
0.500 
0.300 
0.05000
 
0.04595 
0.96898 
130 
0.900 
0.500 
0.400 
0.500 
0.300 
0.05000
 
0.03102 
0.97890 
140 
0.900 
0.500 
0.400 
0.500 
0.300 
0.05000
 
0.02110 
0.98561 
150 
0.900 
0.500 
0.400 
0.500 
0.300 
0.05000
 
0.01439 
 
 
 
Given previous reports indicating a prevalence of suboptimal transtemporal windows in 5% of 
Hispanic20, 5% of Caucasian21, 9% in African American19 and 14% of Asian22 individuals aged <60 
years, we increased our projected sample size by 20% (n=120). In addition, the final sample size 
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was further increased by 20% (n=144) in order to account for an anticipated dropout rate of 20%. 
Consequently, the final study sample was set at 144 individuals.
The sensitivity of TCD against TEE for right-to-left shunt detection has been estimated 90% in a 
recent meta-analysis24. A sample size of 147 patients produces a two-sided 95%CI with a 95%CI 
width equal to 20% when the sample sensitivity is 90% and the prevalence is 30%. After the first 
150 TTE subjects in the Main study are enrolled, the study will continue to enroll up to an additional 
150 subjects that received a TTE or TEE bubble study. 
Primary Safety Endpoint Analysis 
Throughout the study we will collect the incidence of device-related adverse events, which will be 
used to determine the safety of the device within the acute stroke environment. In our EXPEDITE 
study which is also run in the ER for stroke assessment, the device has been designated minimal 
risk by the IRB and we have not had any serious adverse events in over 80 subjects. We will 
continue to closely monitor this metric.
Secondary Safety Endpoint Analysis
Throughout the study we will collect the incidence of procedure-related adverse events related to 
agitated saline injection before and after VM (headache, allergic reactions, new onset neurological 
deficit, ischemic stroke, TIA or pulmonary embolism complicating agitated saline contrast injection, 
etc.) will be closely monitored and prospectively collected18.
ETHICAL CONSIDERATIONS
Investigational Review Board (IRB) Approval
This study will be conducted using an FDA cleared product called the Lucid M1 System, 510k 
K160442. The study will also utilize an investigational device, called the NeuralBot Investigational
System (NB-IS) 510k K180455 with modifications to facilitate workflow improvements in the study. 
The modifications do not introduce additional risks and do not alter existing risks and mitigations. 
All modifications will be tested per NovaSignal’s design controls procedures.
This protocol and the informed consent must be reviewed and approved by the appropriate IRB 
where the study is to be conducted before enrolment of subjects. The NovaSignal and the IRB 
must approve in writing any changes to the protocol that affect the rights, safety, and/or welfare 
of the subjects, or may adversely affect the validity of the study. 
It is the responsibility of the investigator to submit the final version of the protocol with the Informed 
Consent Form (ICF), if required, to an appropriately constituted IRB prior to commencement of 
the study. The Investigator will submit the appropriate documentation if any extension, renewal or 
amendment of the IRB approval must be obtained. In particular, study plan amendments, ICF 
changes or other written information provided to the subject must be approved by the IRB in 
writing, when required. 
Role of NovaSignal
As the study Sponsor of this clinical study, NovaSignal has the overall responsibility for the 
conduct of the study, including assurance that the study meets the regulatory requirements of the 
Food and Drug Administration. NovaSignal will ensure adherence to the regulations as outlined in 
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the Sponsor general duties, selection of investigators, monitoring, maintaining records, and 
submitting reports.
Investigator Responsibilities 
The Principal Investigator (PI) shall be responsible for the day-to-day conduct of the study as well 
as for the safety and well-being of the human subjects involved in the study. The PI also assumes 
overall responsibility and accountability for the study team and for data obtained from each subject 
participating in the study.
The PI shall be responsible for:
1. Obtaining a written IRB approval for the study and subject ICF prior to including any subject 
in this study, as required by local rules and laws.
2. Ensuring that the study is conducted in compliance with IRB requirements including 
conditions which may be imposed by a reviewing IRB.
3. Ensuring compliance with the study plan, applicable laws, and applicable regulations.
4. Obtaining informed consent and privacy authorization for all study subjects prior to subject 
participation (the informed consent and privacy authorization processes may be combined, 
as per usual procedure of the IRB).
5. Collecting all required study data on the Case Report Forms provided.
6. Reviewing and signing CRF pages indicating documents are accurate and complete.
The PI will agree to provide access to the records of all subjects entered into this study, as well 
as all other study documentation. In addition, all records may be subject to inspection by officials 
of US FDA and other regulatory authorities according to local rules and laws. 
The PI should make accurate and adequate progress reports to the IRB at appropriate intervals, 
according to the IRB requirements, when applicable. The PI will inform the IRB of study completion 
or termination within the time period specified by the IRB, when applicable.
The PI is responsible for informing the IRB of any safety issues related to the study as required.
The PI/site must maintain adequate records on all aspects of the study. 
The PI/site must maintain the study records for at least two years after cessation of the study.
Sub-Investigators will be responsible for study activities in coordination with the PI and in 
accordance to the study plan. A Sub-Investigator may assume the responsibility of the PI should 
the PI resign from the study.  
Records Custody
An investigator may withdraw from the study. If the PI withdraws from the study, the responsibility 
of conducting follow-up and maintaining records must be transferred to another responsible party 
within institution (i.e. Sub-I). Notice of transfer must be provided in writing by the PI to NovaSignal
and the IRB when applicable, not later than 10 working days after transfer occurs.
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APPENDIX
Spencer Logarithmic Scale (SLS)10
SLS GRADE
SLS Microembolic Signal (MES) Count
Grade 0
No MES
Grade 1
1 – 10 MES
Grade 2
11 – 30 MES
Grade 3
31 – 100 MES
Grade 4
101 – 300 MES
Grade 5
> 300 MES
International Consensus Criteria (ICC)11
ICC GRADE
ICC Microembolic Signal (MES) Count
Grade 0
No MES
Grade 1
1 – 20 MES
Grade 2
> 20 MES or “shower” appearance
Grade 3
“curtain” appearance of MES
Classification of Potential Causative Mechanism in PFO–Associated Stroke1
RoPE Score
Risk Source
Features
Low
High
Very High
A PFO and a straddling thrombus
Definite
Definite
High
(1) Concomitant pulmonary embolism or deep venous 
thrombosis preceding an index infarct combined with either 
(2a) a PFO and an atrial septal aneurysm or (2b) a large-
shunt PFO
Probable
Highly probable
Medium
Either (1) a PFO and an atrial septal aneurysm or (2) a 
large-shunt PFO
Possible
Probable
Low
A small-shunt PFO without and atrial septal aneurysm
Unlikely
Possible
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REFERENCES 
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Lutsep H, Mojadidi MK, Messé SR, Mas JL, Mattle HP, Meier B, Mahmoud A, Mahmoud AN, 
Nietlispach F, Patel NK, Rhodes JF, Reisman M, Sommer RJ, Sievert H, Søndergaard L, 
Zaman MO, Thaler D, Tobis JM; MSCAI14. Proposal for Updated Nomenclature and 
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Paraskevaidis I, Parissis J, Bogiatzi C, Zompola C, Ellul J, Triantafyllou N, Voumvourakis K, 
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10. Spencer MP, Moehring MA, Jesurum J, Gray WA, Olsen JV, Reisman M. Power M mode 
transcranial doppler for diagnosis of patent foramen ovale and assessing transcatheter 
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11. Jauss M, Zanette E. Detection of right-to-left shunt with ultrasound contrast agent and 
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12. Romero JR, Frey JL, Schwamm LH, et al. Cerebral ischemic events associated with “bubble 
study” for identification of right to left shunts. Stroke 2009;40:2343-2348 
 
13. Hart RG, Diener HC, Coutts SB, Easton JD, Granger CB, O’Donnell MJ, et al.; Cryptogenic 
Stroke/ESUS International Working Group. Embolic strokes of undetermined source: the case 
for a new clinical construct.Lancet Neurol. 2014;13:429–438. 
 
14. Sastry S, Daly, K, Chengodu, T and McCollum, C. Is transcranial Doppler for the detection of 
venous-to-arterial circulation shunts reproducible? Cerebrovasc Dis. 2007; 5-6:424-429. 
 
15. Gentile M, De Vito A, Azzini C, Tamborino C, Casetta I. Adding blood to agitated saline 
significantly improves detection of right-to-left shunt by contrast-transcranial color-coded 
duplex sonography. Ultrasound Med Biol. 2014 Nov;40(11):2637-41. 
 
16. Malhotra K, Katsanos AH, Frogoudaki A, Vrettou A, Ikonomidis I, Tsivgoulis G, Patent 
Foramen Ovale: Risk Factors and Diagnostic Comparison of Transcranial Doppler versus 
Echocardiography for Secondary Stroke Prevention. J Neurosonol Neuroimag. 2019;11:22-33 
 
17. Alexandrov AV, Sloan MA, Tegeler CH, Newell DN, Lumsden A, Garami Z, Levy CR, Wong 
LK, Douville C, Kaps M, Tsivgoulis G; American Society of Neuroimaging Practice Guidelines 
Committee. Practice standards for transcranial Doppler (TCD) ultrasound. Part II. Clinical 
indications and expected outcomes. J Neuroimaging. 2012 Jul;22(3):215-24. 
 
18. Romero JR, Frey JL, Schwamm LH, et al. Cerebral ischemic events associated with “bubble 
study” for identification of right to left shunts. Stroke 2009;40:2343-2348. 
 
19. Tsivgoulis G, Stamboulis E, Sharma VK, Heliopoulos I, Voumvourakis K, Teoh HL, Vadikolias 
K, Triantafyllou N, Chan BP, Vasdekis SN, Piperidou C. Safety of transcranial Doppler 'bubble 
study' for identification of right to left shunts: an international multicentre study. J Neurol 
Neurosurg Psychiatry. 2011Nov;82(11):1206-8. 
 
20. Brunser AM, Silva C, Cárcamo D, Muñoz P, Hoppe A, Olavarría VV, Díaz V, Abarca J. 
Transcranial Doppler in a Hispanic-Mestizo population with neurological diseases: a study of 
sonographic window and its determinants. Brain Behav. 2012 May;2(3):231-6. 
 
21. Marinoni M, Ginanneschi A, Forleo P, Amaducci L. Technical limits in transcranial Doppler 
recording: inadequate acoustic windows. Ultrasound Med Biol. 1997;23(8):1275-7. 
 
22. Itoh T, Matsumoto M, Handa N, Maeda H, Hougaku H, Hashimoto H, Etani H, Tsukamoto Y, 
Kamada T. Rate of successful recording of blood flow signals in the middle cerebral artery 
using transcranial Doppler sonography. Stroke. 1993 Aug;24(8):1192-5. 
 
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23. Saric M, Armour AC, Arnaout MS, et al. Guidelines for the Use of Echocardiography in the 
Evaluation of a Cardiac Source of Embolism. J Am Soc Echocardiogr. 2016;29(1):1‐42. 
doi:10.1016/j.echo.2015.09.011. 
 
24. Katsanos AH, Psaltopoulou T, Sergentanis TN, et al. Transcranial Doppler versus 
transthoracic echocardiography for the detection of patent foramen ovale in patients with 
cryptogenic cerebral ischemia: A systematic review and diagnostic test accuracy meta-
analysis. Ann Neurol. 2016;79(4):625-635 
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