In Vivo Liquid Biopsy for Early Detection of Metastatic Melanoma
Phase: NA
Conditions: Melanoma, Circulating Tumor Cells, Metastatic Melanoma, Diagnosis
Sponsor: Cytoastra

CONFIDENTIAL
 Copyright 2023
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APPROVAL NOTICE
DATE:
15 Nov 2024
TO:
Ekaterina Galanzha, PhD
PROTOCOL:
CytoAstra, LLC, In vivo liquid biopsy for early detection of metastatic 
melanoma (Pro00075507)
APPROVAL DATE:
12 Nov 2024
IRB APPROVED:
Documentation:

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CONFIDENTIAL
 Copyright 2023
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Thank you for continuing to use Advarra IRB to provide oversight for your research project. 
Sincerely,
Luke Gelinas, PhD
Executive Board Chair

 
1
Study Title: In vivo liquid biopsy for early detection of metastatic melanoma  
Proposed dates for data collection:  
Start date: TBD 
Institution/Sponsor: CytoAstra LLC 
I certify that the information provided in this application is complete and correct.  I understand 
that as principal investigator (researcher), I have ultimate responsibility for the conduct of the 
study, adherence to ethical standards, and protection of the rights and welfare of human 
participants. I agree to: (1) conduct the study according to the approved protocol; (2) make no 
changes to the approved study without prior IRB approval; (3) use the approved procedure and 
form(s) for obtaining informed consent; and, (4) promptly report any significant adverse events to 
the IRB within five working days of occurrence. 
 
  
Researcher’s Name 
 
 
 
       
 
 
 
      Date 
 
  
Advisor/Mentor/Supervisor/Sponsor Name   
 
 
                  Date 
 
 

 
2
Table of Contents 
Abbreviations………………………………………………………………………3 
Study Summary (Goal, Hypothesis and Objectives)………………………………5 
Background………………………………………………………………………...6 
Study Design/Procedures Involved ………………………………………………..9 
Study Calendar ……………………………………………………………………13 
Description of Investigational Device and Monitoring Procedure………………...15 
Subject Population and Inclusion/Exclusion Criteria……………………………...16 
Recruitment Methods ……………………………………………………………...18 
Consent Methods …………………………………………………………………. 19 
Potential Risks/Benefits to Subjects or Others……………………………………. 20 
Safety Endpoints …………………………………………………………………...21 
Withdrawal of Subjects …………………………………………………………….22 
Provisions to Monitor the Data to Ensure the Safety of Subjects ………………….23 
Provisions to Protect the Privacy Interests of Subjects …………………………….24 
Subject Compensation/Reimbursement ……………………………………………25 
Data Management and Confidentiality …………………………………………….26 
Conflict of Interest …………………………………………………………………28 
Dissemination of Data ……………………………………………………………..29 
Sharing of Results with Subjects …………………………………………………..30 
Data Analysis and Evaluation Techniques ………………………………………...31 
Data Storage ………………………………………………………………………..34 
Bibliography ………………………………………………………………………..35 
Figures ……………………………………………………………………………...37 
 
 
 

 
3
Abbreviations 
AE 
 
- Adverse event 
AJCC  
- American Joint Committee on Cancer 
CCTRA 
- Cancer clinical trials and regulatory affairs  
cfDNA 
- cell-free DNA 
CT 
 
- Computer tomography 
CTC   
- Circulating tumor cell 
ctDNA  
- circulating tumor DNA 
DNA  
- Deoxyribonucleic acid 
DOB  
- Date of birthday 
FDA  
- Food and Drug Administration 
HIPAA 
- Health insurance portability and accountability act 
Hz 
 
- Hertz 
ID 
 
- Identification 
IDE 
 
- Investigational device exemption 
In vivo  
- Latin for “in a living organism” 
IRB 
 
- Institutional review board 
kHz 
 
- Kilo-hertz 
LLC 
 
- Limited liability company 
MEM  
- Error on mean 
MRI 
 
- Magnetic resonance imaging 
J 
 
- Microjoule 
mL 
 
- Milliliter 
nm 
 
- Nanometer 
ns 
 
- Nanosecond 
NSR 
 
- Non-significant risk 
OS 
 
- Overall survival 
PA 
 
- Photoacoustic 
PAFC  
- Photoacoustic flow cytometry 
PET 
 
- Positron emission tomography 
PFS 
 
- Progression free survival 
PHI 
 
- Protected health information 

 
4
PI 
 
- Principle investigator 
RNA  
- Ribonucleic acid 
SLN 
 
- Sentinel lymph node 
TBD  
- To be determined 
US 
 
- United States 
 
 

 
5
Study Summary (Goal, Hypothesis and Objectives) 
The majority of melanoma deaths are associated with metastasis that are formed by cancer cells 
shedding from the primary tumor, enter the circulation and spread to distant organs via the blood.1-
2 These cells in blood are termed circulating tumor cells (CTCs). Growing  evidence indicates that 
metastasis is an early event in melanoma patients, often occurring even before metastases are 
clinically detectable.1,3 Therefore, detection of CTCs using liquid biopsy assays should be 
considered as effective tools to early diagnose a risk of metastasis progression.1-2, 4-5 Taking into 
account the link of CTCs with metastasis, and thus melanoma aggressiveness, CTCs have an 
advantage over many other biomarkers in identifying patients for early therapeutic intervention at 
a stage when the disease is still curable. This advantage might also help to avoid underdiagnosis, 
which is a major issue in melanoma. Overall, despite CTCs spread occurs early, they are usually 
currently detected late.1 
Currently, the main issue restricting the use of CTCs for early melanoma detection relates to the 
low sensitivity in the detection of CTCs in conventional small blood samples.1,6-7 Clearly, it is 
important to maximize the blood volume available for analysis. It can be achieved by in vivo CTC 
assessment. Toward this goal, in vivo CTC enumeration without labelling in a large blood volume 
using photoacoustic (PA) flow cytometry (PAFC) diagnostic platform was developed by our 
team.6 Clinical testing of this technology revealed a detection sensitivity of 1 CTC per liter of 
blood (~1,000 times better than that of existing assays), with CTCs being detected in 27 (sensitivity 
96.4%) of 28 patients with melanoma and 0 of 19 individuals without cancer (specificity 100%).7 
The Goal of this clinical study is to determine whether a PAFC device called the Cytophone can 
be used for in vivo detection and enumeration of circulating tumor cells (CTCs) in the melanoma 
patients at different disease stages with focus on early diagnosis, early assessment of disease 
recurrence and monitoring of therapy efficiency. It is expected that the detection limit of CTCs 
will be improved at least an order of magnitude compared to the detection limits of existing 
methods.   
We hypothesize that PAFC-based CTC assay in vivo provides earlier, rapid and more accurate 
prognosis of metastasis progression, disease recurrence and therapy efficiency. 
To achieve our goal, we will accomplish the following primary and secondary objectives:    
The primary objectives: 
1. Obtain evidence that positive PAFC-based CTC test indicate a risk of metastasis 
development 
2. Define thresholds of CTC counts that correlate with melanoma recurrence and progression 
of metastatic disease and therapy efficacy 
The secondary objective: 
1. Determine if PAFC diagnoses risk of melanoma metastasis progression and recurrence 
earlier than existing methods.  
 
 

 
6
Background 
Worldwide, more than 350,000 people are diagnosed with melanoma and nearly 60,000 deaths are 
attributed to melanoma annually.8 In the US, melanoma is the fifth most common cancer 
(https://www.cancer.net/cancer-types/melanoma/statistics) resulting in a high mortality rate. 
According to the American Cancer Society (https://www.cancer.org/cancer/melanoma-skin-
cancer/about/key-statistics.html), the estimates for melanoma in the US for 2023 is about 97,610 
new cases, and about 7,990 people are expected to die of melanoma. The main cause of melanoma 
deaths is metastasis.1,8-9 Indeed, the localized primary tumors can be effectively treated, but if 
tumor cells detach from primary tumor, prognosis worsens, and treatment becomes much less 
effective. As an example, the 5-year relative survival rate in patients with "thin melanoma," with 
no detectable metastasis is 99% while it dropped to 32% for patients with metastatic melanomas.9  
Despite remarkable progress which has been made on both the diagnosis and therapy of metastatic 
melanoma, the incidence, recurrence, and death rates remain unacceptably high.9  
It is important that (1) a median delay of early melanoma diagnosis is estimated at approximately 
9 months;8,10 and (2) many people still don't benefit from the newest drugs because of melanoma 
recurrence after initial successful treatment.11 Thus, new diagnostic tools to identify early 
melanoma, especially, its risk to metastasis progression and risk of recurrence in melanoma 
patients are of paramount importance for surveillance and treatment plans. 
Based on our comprehensive literature review, diagnostic challenges that need resolved are 
outlined below.  
Early detection of a melanoma is a major concern for the clinicians because the existing methods 
such as biopsy and non-invasive imaging, which are approved in medical practice, are limited to 
detect early-stage melanoma and risk of its recurrence before the onset of symptomatic metastases 
already exist.1,4,9-11 Among many methods, blood melanoma biomarkers (e.g., CTCs, and ctDNA) 
holds promise to solve diagnostic challenges because CTC detection has potential for pinpointing 
patients who are prone to metastasis.4,12-17 As the evidence, multiple clinical studies has been 
demonstrated that CTC count is associated with risk of disease recurrence and progression free 
survival (PFS) and overall survival (OS).2,5,13,15-17 Furthermore, our preclinical study with mouse 
models showed that melanoma CTCs were detectable soon after formation of primary tumor and 
before histologically detectable metastasis.3,18-19 Together, these data show that CTCs have the 
potential to be used in the early detection and/or diagnosis of recurrence of melanoma. However, 
there is no consensus between physicians on the role of blood tests in melanoma diagnosis and 
follow-up.2,20 This is because there are critical limitations of existing CTC tests. The main 
drawback of all existing CTC assays is their low sensitivity due to the in vitro methodology.1,3,7 
Specifically, examination of a small volume blood sample (≤10 ml) prevents detection of CTCs in 
the other 4,990-ml of a ~5-liter total human blood volume.3,7 Since an early-stage tumor is 
expected to produce very small number of CTCs (1–3 CTC or less/~5 billion blood cells, current 
methods lack early CTCs that prevents detection/prognosis of early metastasis.1,3,6-7,18 Other 
limitations of CTC methods include low throughput, which require many hours to assess a blood 
sample in vitro; and multiple sample-processing steps, which result in a 60% to 97% loss of CTCs.1 
In recent years, the efforts have been made to develop new liquid biomarkers such as cell-free 
nucleic acids including cell-free DNA (cfDNA), ctDNA and cell-free microRNA.2,4,17, It has been 
found that some CTC-derived mRNA and ctDNA were indicative of clinical outcomes for patients 
with stage IV melanoma2 but usefulness of these assays is limited by unknown the origin of 

 
7
detected nucleic acids. From the use of existing tests, it is not clear whether the ctDNA is actively 
released from live cells or derived from cells that have undergone apoptosis/necrosis.2 
Furthermore, to distinguish ctDNA from cfDNA, specific somatic mutations in the melanoma 
tumor must first be identified, but this is sometimes challenging in obtaining sufficient tumor 
material for genetic investigation within the clinical setting.13,21-22  This is  because some tumors 
can only be accessed through invasive procedures that provide insufficient genotyping material. 
To fill this a gap in early melanoma diagnosis, designing more sensitive and accurate CTC 
diagnostic tool is in high demand. The ideal method should be highly specific and sensitive to 
identify patients who are at high-risk of metastasis progression and recurrence after treatment, 
provide treatment outcomes to personalize systemic treatment, provide prognostic data before, 
during and after treatment, and monitor response to treatment. Based on our successful preclinical 
and recent clinical studies,3,7,18-19 we demonstrated that the PAFC diagnostic platform could be 
part of the solution (Fig.1). In PAFC, the principle of PA detection, coupled with using melanin 
as an intrinsic marker, to which PA methods are very sensitive (Fig. 2). In particular, our clinical 
trials with PAFC technology revealed a detection limit down to 1 CTC per a liter of blood (~1,000 
times better than that of existing CTC assays), with CTCs being detected in 27 (i.e., sensitivity 
96.4%) of 28 patients with melanoma and 0 of 19 individuals without cancer (i.e., specificity, 
100%) (Fig. 3).7 Based on the obtained data, PAFC advantages include: 
• In vivo monitoring of circulating blood to achieve assessment of CTCs in up to one liter of 
blood volume. 
• High throughout because initial diagnostic results such as POSITIVE/NEGATIVE CTC 
test will be obtained during the monitoring procedure without additional processing steps. 
• Recorded and software-treated results will be used to receive extended diagnostic 
information on CTC including CTC count per a time unit. 
• Preventing the potential loss of CTCs because The Cytophone’s diagnosis does not require 
multi-step processing procedures. 
• High specificity and sensitivity of PAFC device in detection of melanoma CTCs. 
• Reducing toxicity concern because detection procedure is noninvasive and label free: well-
known high PA contrasts of intrinsic melanin in CTCs provides label-free diagnosis 
without injection of any reagents in blood circulation, and in vivo approach prevents blood 
collection and makes procedure completely noninvasive. 
We anticipated that CTCs detected by PAFC will be the first indicators of the early melanoma cell 
spreading through blood system with the risk of metastasis development. The Cytophone would 
enable monitoring for this process in a noninvasive manner and have the potential to be applied as 
a tool for the early detection of metastatically aggressive melanomas. Also, it can support 
clinicians in making individual (personalized, tailored) plans of treatments and determining who 
should receive adjuvant therapy (e.g., immunotherapy) resulting in personalized medicine. This 
decision can help to prevent metastatic progression by early initiation of personalized therapies 
and minimize the number of patients exposed to treatment-related toxicities that eventually 
improve patient survival. In addition, PAFC detection procedure would not only improve 
prognosis and therapy but would ultimately reduce the costs by preventing unnecessary biopsies, 
screening, and reducing treatment costs. The additional benefits of the Cytophone technology 
include the expected access to patients (as a mobile-like device) with very low to no challenges in 
physician training. It should be emphasized that PAFC is not intended to replace the well-

 
8
established clinical diagnoses. We anticipated that PAFC would supplement histopathological and 
imaging diagnosis. 
In future, we expect that PAFC technology would have significant advances in using CTCs as 
excellent marker for screening population to identify asymptomatic individuals with high-risk of 
metastatic melanoma. 
 
 

 
9
Study Design / Procedures Involved 
Start Date: TBD 
End Date: TBD 
This study will be conducted the Cytophone device for subjects with diagnosed melanoma. 
Participants need to be 18 years of age or older. The study is not intended to change routine 
diagnosis and therapy of melanoma patients. Eligible participants will be given the Cytophone’s 
procedure in person after signing the Informed Consent Form and verification of the subject ID, 
date of birthday (DOB) and name. The blood samples will be collected before a the Cytophone’s 
procedure to detect CTCs and ctDNA using conventional methods in vitro. Blood samples will be 
obtained in the laboratory by a nurse and transferred to the companies for testing melanoma in 
vitro: Menarini (CTC test) and Billion to One (ctDNA test). Results will be provided by the 
companies in a few weeks.  
It is anticipated that PAFC diagnostic procedure will take approximately thirty (30) minutes 
including ultrasound imaging of an examined vessel, photo of a skin area above the vessel and 
quick navigation of PAFC probe on the vessel (Fig. 3, top). At early disease stage, the diagnostic 
procedure can be increased to one hour to test larger blood volume and get more statistical data.   
Subjects will come into the assigned clinical room for up to 5 visits. A withdrawal visit will be 
scheduled as needed. A post observational (medical records, OS and PFS) study design will be 
implemented to define progression and recurrence of metastasis. 
Participants will be screened for any contraindications (physical exam and lab work) to taking the 
included investigational procedure and evaluated per protocol inclusion/exclusion criteria. The 
risk to subjects in this trial will be minimal by compliance with the inclusion/exclusion criteria, 
regular clinical monitoring, avoidance of unnecessary procedures, and adherence to investigator 
guidance regarding specific safety areas. If, during the study, a female participant becomes 
pregnant or decides to attempt to become pregnant, then she must stop the study and undergo the 
rapid exclusion procedure. Female patients must not be breastfeeding or pregnant (as confirmed 
by serum pregnancy test) at the time of study entry and must agree to undergo serum pregnancy 
testing throughout the study at each clinic visit. In addition, a serum pregnancy testing should be 
conducted in case of an unexpected delay of menorrhea. 
Location of the study TBD   
Groups of the study. All study participants will be blinded from their results. There will be four 
groups to the study. Subjects will be assigned to study cohorts based on the AJCC Melanoma of 
the Skin Disease Staging (8
th edition) as documented at the time of consent based on current or 
previous disease status. 
1. Group 1: The Cytophone testing of healthy subjects (30 individuals). This group will 
consist of thirty healthy subjects with all of light skin tones and ten of whom will be of 
dark skin tones according to the Fitzpatrick classification scale. Subjects will have two 
visits: one visit for checking eligibility + signing a consent form and one visit for The 
Cytophone’s testing. The obtained data will be used to address the calibration goal. Healthy 
subjects will be recruited by the assigned clinics based on the willing to volunteer for this 
group. Clinicians or assigned clinical coordinator will inform the potential subject of the 
study and refer them to the study team for more information.  

 
10
2. Group 2: The Cytophone testing of 50 subjects who have been pathologically diagnosed 
with Stage 0-I melanoma.  The initial PAFC testing will occur within 30 days of diagnosis. 
Subjects will have 4 visits. The first visit will include checking eligibility + signing a 
consent form, blood draw and the Cytophone’s testing. The follow-up three visits (visits 2, 
3 and 4) will include the Cytophone’s testing. Group 2 participants will be asked to provide 
their ID number in order to link their in vivo CTC count with medical records including 
biopsy. The obtained data will be used to link the presence of CTCs in blood circulation or 
increase in CTC count with metastatic disease initiation.  
3. Group 3: The Cytophone testing of 50 subjects who have been pathologically diagnosed 
with Stage II melanoma. The initial PAFC testing will occur within 30 days of diagnosis. 
Subjects will have 4 visits. The first visit will include checking eligibility + signing a 
consent form, blood draw and the Cytophone’s testing. The follow-up three visits (visits 2, 
3 and 4) will include the Cytophone’s testing. An increase in CTC count will possibly 
demonstrate an early sign of initiation of disease progression. Group 3 participants will be 
asked to provide their ID number in order to link their in vivo CTC count with medical 
records including dermoscopy, biopsy, sentinel lymph node (SLN) status, CT, MRI, PET 
and others. The results will be used to analyze the correlation between CTC count and 
metastatic disease initiation/progression and further recurrence induced by metastasis-
originated CTCs.  
4. Group 4: The Cytophone testing of 50 subjects who have advanced-stage melanoma (stages 
III - IV) with metastatic disease. Subjects will have 4 visits. The first visit will include 
checking eligibility + signing a consent form, blood draw and the Cytophone’s testing. The 
follow-up three visits (visits 2, 3 and 4) will include the Cytophone’s testing. Dynamic of 
CTC counts will be defined over disease progression and treatment to link CTC counts, 
metastatic disease progression and therapy efficiency. Group 4 participants will be asked 
to provide their ID number in order to link their in vivo CTC count with medical records. 
Visits. The study will last less than two years and will includes 4 visits, and 12 months follow-up 
observation of medical records, OS and PFS after 4th visit. 
Visit 1 (90 minutes) – at any time in Group 1, after the diagnosis of melanoma in Groups 2-4. The 
visit will start with checking inclusion and exclusion criteria, confirming eligibility, and obtaining 
the informed consent. Only subjects who are eligible and have signed off on the consent form 
will participate in this study. Next, the visit will involve blood collection, ultrasound imaging, 
adjustment PAFC laser beam on the vessel and PAFC monitoring. Also, information on possible 
adverse events and review of medications and diagnostic tests will be collected, and vital signs 
(temperature, pulse, blood pressure) will be measured. A pregnancy test will be done if the subject 
is a female of childbearing potential. 
Visit 2 (60 minutes) – in ~90 days after first visit (Groups 2-4). The visit will involve ultrasound 
imaging, adjustment PAFC laser beam on the vessel and PAFC monitoring. Also, information on 
possible adverse events and review of medications and diagnostic tests will be collected, and vital 
signs (temperature, pulse, blood pressure) will be measured. A pregnancy test will be done if the 
subject is a female of childbearing potential. 
Visit 3 (60 minutes) – in ~90 days after second visit (Groups 2-4). The visit will involve ultrasound 
imaging, adjustment PAFC laser beam on the vessel and PAFC monitoring. Also, information on 
possible adverse events and review of medications and diagnostic tests will be collected, and vital 

 
11
signs (temperature, pulse, blood pressure) will be measured. A pregnancy test will be done if the 
subject is a female of childbearing potential. 
Visit 4 (60 minutes) – in ~90 days after third visit (Groups 2-4). The visit will involve ultrasound 
imaging, adjustment PAFC laser beam on the vessel and PAFC monitoring. Also, information on 
possible adverse events and review of medications and diagnostic tests will be collected, and vital 
signs (temperature, pulse, blood pressure) will be measured. A pregnancy test will be done if the 
subject is a female of childbearing potential. 
Follow-up observation (no visit requires) – in 12 months after 4th visit (Groups 2-4). 
Procedures. 
Investigational PAFC Procedure. At the beginning of each visit, for approximately a few minutes, 
the investigator will adjust the position of laser beam on the vessel. The investigator will use near-
infrared viewer and skin marker.  After completion, the Cytophone monitoring of this vessel will 
be conducted approximately 30 minutes and one hour if needed. The section below provides more 
details of The Cytophone device and related procedures. 
Standard of care procedures 
Specimen Handling (Groups 2-4).  
A blood draw with a volume of 25 mL will occur in the blood laboratory at the site of melanoma 
testing via venipuncture standard practices. The sample will be coded using the study subject ID. 
A 10 mL will be transported to Menarini Silicon Biosystems, Inc. laboratory for detection of CTCs 
in vitro. Another 10 mL will be delivered to Billion to One for ctDNA testing. Pulse, temperature, 
height/weight, blood pressure, and pulse will be measured in the sitting position for all subjects.  
Pregnancy testing: serum pregnancy tests will be carried out for women of child-bearing potential.  
Collected data. 
Demographic data, diagnosis, medical records, and medications will be collected at the first visit 
(pre-visit). Medical records, and medications will be continuing to monitor over a period of 4 clinic 
visits (~ 9 months) and a post-12-month observation.  
The Cytophone records will be use to: 
• Collect background signals in vivo and in vitro to estimate rate of false positive signals 
(Croup 1) 
• Calculate number of PA signals per a time unit that are associated with single CTCs and 
CTC clusters (Groups 2-4) 
At the end of the monitoring and observation period, the investigators will analyze the results and 
determine if the CTC counts correlate with metastatic disease progression and may be used as 
diagnostic and/or prognostic criterium of aggressiveness of early melanoma, initiation/progression 
of metastasis, initiation/progression of recurrence and assessment of therapy efficacy.  
Photographing Participants. 
Participants will be asked a yes or no question on the informed consent as to whether they agree 
or not to allow the research staff to take an ultrasound images and photo during testing for 
presentation purposes ONLY. They will be no identifying facial pictures taken. 

 
12
 
 

 
13
Study Calendar 
 
Visit 1; 
Day 0 
 
Visit 2; 
Month 3 
( 14 Days) 
Visit 3; 
Month 6 
( 14 
Days) 
Visit 4; 
Month 9 
( 14 
Days) 
Observation of 
medical 
records, OS and 
PFS; Month 24 
( 14 Days) 
Group 1 
Informed consent 
X 
 
 
 
 
Inclusion/Exclusion 
criteria 
X 
 
 
 
 
Vital 
signs/pregnancy 
test 
X 
 
 
 
 
PAFC 
X 
 
 
 
 
Photography 
X 
 
 
 
 
Blood sampling 
 
 
 
 
 
Review 
AEs 
/ 
Medications 
/ 
Medical records 
X 
 
 
 
 
Group 2-4 
Informed consent 
X 
 
 
 
 
Inclusion/Exclusion 
criteria 
X 
 
 
 
 
Vital 
signs/pregnancy 
test 
X 
X 
X 
X 
 
PAFC 
X 
X 
X 
X 
 
Photography 
X 
X 
X 
 
 
Blood sampling 
X 
 
 
 
 

 
14
 
 
 
Review 
AEs 
/ 
Medications 
/ 
Medical records 
X 
X 
X 
X 
X 

 
15
Description of New Device and monitoring procedure 
The Cytophone device is based on a noninvasive, label-free PAFC diagnostic platform (Fig. 2). 
The Cytophone will be used to monitor in vivo (1) healthy volunteers to optimize The Cytophone’s 
parameters and estimate false signal rates, and (2) melanoma patients to identify the presence of 
melanoma CTCs and their clusters (aggregates, clots), and to enumerate them per a time unit.  
The PAFC/The Cytophone clinical prototype incorporates pulsed two pulse lasers, optical system 
for delivery of laser light to the skin, ultrasound transducer array and detection electronics for 
acquisition of acoustic waves from CTCs (Fig. 4). Parameters of lasers:   
 
C-Wedge 1064 (Bright Solutions, Cura Carpignano (PV), Italy): 
Wavelength,1064 nm; pulse width, 0.6 ns; pulse rate: 1 kHz; pulse energy, 240 µJ.  
 
C-Wedge 18901 (Bright Solutions, Cura Carpignano (PV), Italy): 
Wavelength, 770 nm; pulse width 3 ns; pulse rate, 1 kHz; pulse energy, 240 µJ. 
The lasers complement each other when in use, if there is a failure or downtime with one of the 
lasers, then the operational laser can still be used monitoring patients. 
Detection of acoustic waves from CTCs is performing by focused ultrasound transducer array 
placed in an acoustic contact with the skin of the subject with ultrasound gel. The signals from the 
transducers are amplified with 16 channel amplifier (Fig. 4), and then digitized by a 12-bit, 500 
MSPS high speed board digitizer (ATS9350, AlazarTech, Canada) and transfer to data recording 
system using a laptop. The PAFC platform is assembled on a cart. The laser body, computer, and 
all the accessories are self-contained within the cart. Internal components of the setup are not 
accessible to the subject. The top of the cart contains the PAFC probe on a thin optical plate.  A 
manual stage is used to provide positioning of the probe over the vein of the backhand, wrist or 
forearm. Delivery of the laser light to the probe is performed by an optical fiber or by lens optics. 
Positions of the ultrasound transducer and optical fiber or lens will be fixed after a short initial 
probe alignment procedure.  
Subjects will receive PAFC procedure while seated in a chair. The subject’s hand will be placed 
on a customized surface, with a PAFC probe placed over a selected vein. Acoustic contact between 
the skin and ultrasound transducer will be provided by a gel. Flexible Velcro strips is used to 
provide gentle fixation of the hand during PAFC monitoring. PAFC will be used to monitor blood 
flow during 30 min. If necessary (e.g., due to signal instability or low signal levels) this procedure 
will be repeated on a different blood vessel(s) up to an additional 30 minutes. PA measurements 
will be performed the entire time the subject holds the hand under the probe. The position of the 
probe over the vein will be monitored, if necessary, by comparing the signals from blood vessel to 
that from surrounding tissues and may require adjustment during the experiment. 
While the device is contained on a cart, it remains in one location, except when maintenance 
requires moving it to the company. If the device is moved for maintenance, it is recalibrated after 
returning it to the clinical site.   
 
 

 
16
Subject Population and Inclusion/Exclusion Criteria 
Study Population: To achieve our goal, we expect to enroll 180 participants (18-80 years old) 
during the proposed timeframe. Males and females will be eligible for this study. No exclusions 
will be made regarding ethnicity. The target is to enroll 20-25% of subjects with dark skin.  
Prior to coming in for the Cytophone’s testing, subjects will sign the consent and complete a 
medical history questionnaire. 
Group 1: 30 healthy subjects, twenty of whom will be of light skin tones and ten of which will be 
of dark skin tones according to the Fitzpatrick classification scale.  
Groups 2-4: 150 subjects (50 subjects per a group) with melanoma will be assigned to study based 
on the AJCC Melanoma of the Skin Disease Staging (8
th edition) as documented at the time of 
consent based on current or previous disease status. 
All participants will receive 4 PAFC procedures over ~9 months. A blood sample will be taken for 
in vitro CTC and ctDNA tests.  
Inclusion and Exclusion Criteria  
Inclusion Criteria for Group 1 (Healthy Subjects):  
• Aged 18–80 years, inclusive 
• Free of concomitant or previous malignancies, as deemed by the Principal Investigator, is 
not contraindicated with any past medical history of the participant 
• Participants having provided informed consent with signature on informed consent form: 
the informed consent process should be complete with full discussion of all requirements 
and possible risks. 
• Must be able to sit for up to 60 minutes  
Inclusion Criteria for Groups 2-4 (Melanoma Patients):  
• Aged 18–80 years, inclusive 
• Histological documented diagnosis of melanoma  
• Participants having provided informed consent with signature on informed consent form: 
the informed consent process should be complete with full discussion of all requirements 
and possible risks. 
• Must be able to sit for up to 60 minutes  
Exclusion Criteria:  
• Unable to provide informed consent to participate in the study, such as a mental condition 
rendering the participant unable to understand the nature, scope, and possible consequences 
of the study 
• Clinically relevant cardiovascular, hepatic, neurological (e.g., evidence of organic brain 
syndrome), endocrine, or other major systemic disease making implementation of the 
protocol or interpretation of the study results difficult or that would put the participant at 
risk by participating in the study 
• Persistent significant or severe infection, either acute or chronic 
• Pregnant or breast-feeding women or those who plan to become pregnant during the study 

 
17
• Women of childbearing potential not protected by effective contraceptive method of birth 
control and/or who are unwilling or unable to be tested for pregnancy. 
• Any known history of severe preexisting constipation 
Research staff will review this list of exclusion criteria with each participant at the time of consent 
in order to identify eligibility. 
 
 

 
18
Recruitment Methods 
The study and recruitment information will be also available to all interested participants on the 
CytoAstra.  
Investigator contact information will be included in all advertisement materials for additional 
questions or concerns regarding the study. 
Subjects who are interested will be asked to contact the study team to complete a prescreening. 
Research staff has been trained on enrollment of patients and Cytophone operation. 
The PI and research assistants will be responsible for recruitment, retention and coordination of 
subjects.  
After signing the IRB-approved informed consent form, research subjects will be registered on the 
clinical site performing the Cytophone test. At that time, a registration number will be provided to 
the Principal Investigator and research staff. The initial procedure must be conducted within 90 
days of consent, or the subject will be withdrawn from the study and will need to re-consent in 
order to participate. Subjects who are withdrawn from the study due to delay of procedure may be 
replaced.  
All these forms clearly indicate the participation in the study is voluntary. 
 
 

 
19
Consent Methods 
The informed consent form will be presented to participants at the first visit. The consent process 
will occur in a private room with the research nurse. A research member will explain the informed 
consent form for each potential participant. All participants will be given the option to ask 
questions, they will be given ample time to consider participation and the benefits and risks 
associated with that participation. The participants will be asked to sign the informed consent upon 
agreement of the terms and conditions associated with the study. Participants will also indicate 
that they have read the consent form and agree to participate by selecting ‘yes’ on the consent form 
page.  
Participants can withdraw from the study at any time without any consequences. 
Signed consent documents returned via fax, email, or mail should be accompanied by copies of 
official documentation identifying the potential subject and witness. Consent documents returned 
by any of these methods will be signed/dated by study staff with the current date. The consent 
process will be documented in the medical record. The original informed consent will be filed with 
the subject file. Each participant will be given a copy of the informed consent. 
 
 

 
20
Potential Risk/Benefits to Subjects or Others 
Risks.  
Minimal risks are expected because the Cytophone (PAFC) device with the same lasers and the 
same parameters was approved by the IRB committee at the University of Arkansas for Medical 
Science (Protocol #133965) and the FDA classified this device as a Non-Significant Risk (NSR) 
medical device with the Investigational Device Exemption (IDE). Moreover, clinical testing of 
melanoma patients in vivo using PAFC with the same parameters revealed no adverse effects.7 
Thus, The Cytophone is regarded as safe but may cause: 
• 
Local skin irritation and redness as well as burning, tingling, itching, stinging or 
localized pain sensations; careful monitoring of skin changes will be provided and local 
treatment including ice pack, skin lotion or topical steroid will be provided as needed.
 
• 
Discomfort during sitting for the time required to complete the procedure; the 
participant may request that the procedure be stopped at any time.
 
Risks associated with blood draws may include bruising, minor pain and rare infection at the 
puncture site. Experienced personnel will perform this procedure using approved techniques. 
Pressure and dressings will be used to minimize pain, bruising and infection. 
There is also a minimal risk associated with loss of confidentiality or anonymity. To minimize this 
risk, participants’ identity will be kept confidential at all times by the research team. Subjects will 
be assigned a study number unrelated to their medical record number or other personally 
identifying information. The obtained research data will be associated with the subject’s number 
only. The key to the participant identity will be kept in a locked file cabinet behind a locked door 
that only the research team will have access to. Signed consent forms will also be kept in this 
locked file cabinet but separate from the data.  
No risks to others are anticipated. 
Benefits. 
While there are no direct benefits to the participants in this study, results may help practitioners to 
better understand the Cytophone (PAFC) diagnosis and use it in future as an advanced test for 
early melanoma diagnosis, early detection of disease recurrence, monitoring of therapy efficiency, 
and potentially melanoma screening.  
 
 

 
21
Safety Endpoints 
If any of the participants reveal procedure-related side effects or Adverse Events (AEs), the subject 
will be withdrawn. If three people are withdrawn for that reason, the study will be stopped. 
 
 

 
22
Withdrawal of Subjects 
Subjects will be withdrawn from the study if: 
1. The Investigator considers it would be in the best interest of the subject (e.g., AEs, 
intercurrent illness or onset of new clinically significant condition). 
2. Female subject becomes pregnant. 
3. There is a protocol violation which increases the risk to the subject. 
4. Subject withdraws consent or is lost to follow-up. 
The subject will be contacted immediately to schedule a withdrawal visit. If the subject cannot be 
reached on first attempt, then a second and third attempts to contact the subject via phone and 
email will be made.  
If and when a subject withdraws from the study for any reason, all efforts will be made to complete 
and follow the participant through observations as thoroughly as possible. A final evaluation will 
be attempted in those willing to participate.  
 
 

 
23
Provisions to Monitor the Data to Ensure the Safety of Subjects 
AEs and safety labs will be reviewed at each visit by the PI. Additionally, subjects will be 
instructed to call the study team to report new symptoms/AEs. These AEs will be recorded in the 
subject’s binder on a cumulative AE log. If a serious AE is unresolved when a subject permanently 
discontinues the study, the subject will be followed until the AE resolves, or the clinical course is 
stabilized.  
An independent safety monitoring committee involving a physician (James Suen, MD), engineer 
(Aayire Yadem, PhD) and bio-statistician (TBD) will review safety labs and AEs after the first 5 
subjects are enrolled and then after the 50th and 100th subjects are enrolled. He/she will notify the 
study team if he determines that the study must stop for safety concerns and will be charged with 
the responsibility of determining if the trial needs to be stopped based on lab criteria and data from 
any unexpected AEs. These unexpected AEs will be promptly reported to the independent safety 
monitoring physician.  
The whole study will be stopped in cases where serious AEs are experienced by 3 or more subjects 
and are not deemed unrelated to participation in the study by the PI or the independent safety 
monitoring committee. Any AE will be immediately communicated to the PI in writing notifying 
of the event(s). 
 
 

 
24
Provisions to Protect the Privacy Interests of Subjects 
All subject interactions will occur in a private room or lab setting. Subjects will be introduced to 
the various study team members by the PI or study coordinator with whom they’ve already 
established a relationship through prescreening or the consent process.  
The research team will obtain consent prior to receiving any information about subjects.  
 
 

 
25
Subject Compensation/Reimbursement 
There could be compensation for participating in this study. The compensation would 
be paid on a per visit basis, and only paid once each visit is completed for the study. 
 
 

 
26
Data Management and Confidentiality  
Study data will be submitted according to protocol requirements for all subjects registered.  
An EDC, Electronic Data Capture system, will be in place for use by the study coordinator and 
their team.   All documents that can be stored in the EDC will be entered and secured.  All data 
will be stored electronically in the EDC or some other secure storage device.   Any external storage 
devices will be encrypted and under the control and supervision of the research team.  Any hard 
or non-electronic records will be stored securely by the research team.  Both electronic and hard 
copies will be secured only allowing access by the research staff.  
Electronic data will be entered into a password protected file. Cytophone’s records and blood 
samples will be coded and labeled with study subject’s number. Subject initials, age, or any 
possibly identifying information will not be included in the code. A code link will be kept in a 
password protected database separate from the blinded codes. This de-identified information (only 
using assigned study numbers) will be kept within the password protected server.  
All data will be stored for a minimum of 5 years after the study is closed with the IRB. Hard copy 
data will be destroyed via secure shredding per HIPAA standards. Electronic data will also be 
permanently deleted. Confidential data and PHI will not be disclosed outside of the study team 
except as required by law or as allowed by the consent (e.g., with the approved IRB or those who 
monitor the study).  
Participants will be asked a yes or no question on the informed consent as to whether they agree 
or not to allow the research staff to take a photo during testing for presentation purposes ONLY. 
Care will be taken to block out identifiable characteristics in the photo such as the participants face 
if they are willing to allow a photo taken during testing.  
Upon collecting the data, the results will be analyzed and documented. All data (completed 
instrument tools, observation notes, and tape recordings) will be kept confidential. Original data 
will be stored in a locked cabinet in the researcher's office. 
Specifically, baseline and demographic characteristics will be summarized by standard descriptive 
summaries (e.g., means and standard deviations for continuous variables such as age and 
percentages for categorical variables such as gender). Non-parametric analyses will be used in 
situations where the variables are not normally distributed. 
CTC counts per a time unit in vivo and in vitro as well as ctDNA concentration at Day 0, Month 
3 or after surgery, Month 6, Month 9 and Month 12 will be calculated and averaged with 
coefficients of variation. The increasing trend of CTC counts will be estimated and correlated with 
other signs of metastasis initiation/progression. 
All subjects entered into the study at the first visit will be included in the safety analysis. 
The frequencies of AEs by type, body system and severity will be summarized. Severe AEs 
will be described in detail. 
Identifiers collected will be entered directly into a prescreening form, which is password protected. 
If the potential subject fails prescreening, identifiers will not be collected. If the potential subject 
declines to participate, identifiers will be destroyed upon declination. If the potential subject signs 
consent, their name and email address will be copied to a screening log in and removed from the 
prescreening form. Any remaining information on the prescreening form will be permanently 
deleted from the system when prescreening closes. PHI will not be reused/disclosed to any other 

 
27
person or entity except as required by law, for authorized oversight of the research, or for other 
research which use/disclosure of PHI would be permitted by the HIPAA privacy regulations. 
Identifiable information collected during prescreening (names, email addresses) will only be 
collected if the potential subject meets the eligibility criteria. Health information will not be 
recorded during prescreening but will be reviewed to confirm eligibility.  
 
 

 
28
Conflict of Interest 
Dr. Galanzha has a proprietary interest in the device, which is associated with CytoAstra, LLC as 
she is an inventor of PAFC (Cytophone), that have obtained US Patents, 11154360 and 11259705. 
She is also employed by CytoAstra. To prevent any COI concerns, Dr. Galanzha will NOT have 
direct contact with human subjects on this study at any time or will not try to recruit subjects for 
this study.  
Drs. Yulian Menyaev, Safi Ullah, and Aayire Yadem have a proprietary interest in the device 
associated with CytoAstra, LLC.   They are currently employed by CytoAstra.   They will serve 
as operators of the device and will only be referred a subject ID when administering the device.  
Communication will only be limited with the subject to successfully administer the device. 
 
 

 
29
Dissemination of Data 
Results of this study may be used for presentations, posters or publications. The publications will 
not contain any identifiable information that could be linked to a subject and will be reviewed by 
the Sponsor prior to publication. 
 
 

 
30
Sharing of Results with Subjects 
Results will not be shared with subjects. 
 
 

 
31
Data Analysis and Evaluation Techniques 
This study will help to develop protocol and standards for in diagnostic CTC test in vivo for using 
in routine clinical practice for melanoma patients. The obtained results will be published in the 
peer-reviewed journals, presented at the conferences and used in gaining broader approval of the 
technology by regulatory agencies. The data analysis will be conducted as outlined below. 
Analysis plan for Group1 (healthy subjects): The study will provide data for the optimization of 
technical parameters of PA probes that do not require special statistical consideration. Specifically, 
it will include medium types for acoustic coupling of transducer and skin, method of visualization 
of laser beam on the skin and vessels, mechanical holder for spatial fixation of PA probe near skin, 
and signals from blood vessels. 
We will define beforehand a window of equivalence with upper and lower limits of equivalence. 
For example, if we want the two signal amplitudes to be similar to within ±30%, then the window 
of equivalence would have an “upper limit of equivalence” at +30% and a “lower limit of 
equivalence” at –30%. Measure 20 blood vessels in the dorsal side of the hands of 3-5 individuals. 
We can use both hands from the same person. On each vessel, we will use both lasers in second 
mode for vessels only (background signals). Then we will calculate the difference in signal 
amplitudes from each laser. We will express the difference as a percentage relative to skin 
background without visible vessel as well as the percentage relative to Laser 1, or the percentage 
relative to 0.5*(Laser 1 + Laser 2). Then we will calculate the mean of the percent differences and 
calculate the 90% confidence interval on that mean. If both ends of the 90% confidence interval 
fall inside the window of equivalence (i.e., if both ends fall in between the upper and lower limits 
of equivalence), then you will have established that the two lasers give background signals from 
vessels only whose signal amplitudes are equivalent to within ±30%. 
PA signals from the vessels, PA signals from the surrounding tissues, and PA contrasts will be 
displayed as dot plots; subjects will be grouped according to skin pigmentation. PA signals and 
contrasts also will be displayed as scatterplots versus vessel diameter, color-coded by subject. Data 
will be reported by vessel location and skin pigmentation as the mean, standard deviation (SD), 
median, quartiles, and range. Additionally, each PA contrast will be evaluated for whether it is 
“usable” vessel location, then humans. In the secondary post-hoc analysis, we will conduct 
pairwise comparisons of the skin pigmentations, and likewise of the body locations, using a two-
sided 5% alpha despite the multiple comparisons in order not to inflate Type II error. The vessel 
diameter will be added to the mixed model as a continuous covariate to determine whether larger 
PA contrasts are associated with larger vessel diameters, and whether including vessel diameter 
reduces the width of the 90% confidence intervals around the group means. 
The defined parameters will be used for further statistical considerations in Groups 2-4. 
Analysis plan for Groups 2-4 (melanoma patients): For each melanoma subject, the results of in 
vitro assay will be classified as a detection or non-detection of CTCs; if the assay result indicates 
that CTCs were detected, then CTC concentration will also be estimated and classified as above or 
below the effective lower limit of the standard curve’s accuracy. On the same melanoma subject, 
the in vivo Cytophone’s results will be classified as a detection or non-detection of CTCs; if CTCs 
are detected, then the CTC counts per minute will be converted into a CTC concentration using 
the blood-flow rate obtained from Doppler ultrasonography on the same vessel region. The 
discordance rate in CTC detection between in vivo and in vitro method will be determined using 
paired-data contingency tables. Discordances will then be tested at 5% alpha via one-sided 

 
32
McNemar’s tests for significant imbalance favoring successful detection by the in vivo method. 
Additionally, whenever the in vivo Cytophone detects CTCs but the in vitro assay does not, the in 
vivo CTC concentration will be converted via the Poisson distribution into a non-detection 
probability for the in vitro assay based on the reaction volume utilized by the in vitro method. 
Finally, whenever CTCs are detected by both in vivo and in vitro method, the paired difference in 
CTC concentrations between in vivo and in vitro method will be calculated and summarized using 
the number, mean, and SD. 
We will define beforehand a window of equivalence with upper and lower limits of equivalence. 
For example, if we want the two signal amplitudes from CTCs to be similar to within ±50%, then 
the window of equivalence would have an “upper limit of equivalence” at +50% and a “lower limit 
of equivalence” at –50%. We will measure CTCs in  blood vessels in the dorsal side of the hands 
of 5-7 individuals. On each vessel, we will use both lasers. Then we will calculate the difference 
in signal amplitudes from each laser. We will express the difference as a percentage relative to 
vessel background as well as the percentage relative to Laser 1, or the percentage relative to 
0.5*(Laser 1 + calculate the 90% confidence interval on that mean). If both ends of the 90% 
confidence interval fall inside the window of equivalence (i.e., if both ends fall in between the 
upper and lower limits of equivalence), then you will have established that the two lasers give the 
signals from CTCs only whose signal amplitudes are equivalent to within ±50%. 
For in vitro assay, the relevant standard curve will be used to estimate a CTC concentration in the 
blood of each melanoma subject. On the same subject, the CTC counts per minute obtained from 
in vivo Cytophone monitoring will be converted into a CTC concentration using the blood-flow 
rate obtained from Doppler ultrasonography on the same vein region. Data will be transformed to 
their base-10 logarithms both to reduce heteroscedasticity and to facilitate inference on fold-
change differences. Agreement between in vivo and in vitro methods will be assessed using the 
methods of Bland and Altman. Specifically, for the in vivo method and in vitro method, the average 
and difference in CTC counts between methods will be calculated for each subject and plotted in 
a Bland-Altman plot. The 95% limits of agreement between in vivo PAFC and in vitro method 
will be calculated from the difference in CTC counts as the mean ±2 standard deviations. The mean 
difference in CTC counts between in vivo and in vitro method will be reported along with a two-
sided 95% confidence interval. The mean differences of 5-fold or less will be considered good 
concordance between in vivo and the in vitro methods, whereas mean differences of 10-fold or more 
will be considered cause for concern. 
Sample size considerations: For the primary analysis of the Group 1, we will use a mixed- models 
repeated-measures approach to estimate the skin-pigmentation group means and symmetric two- 
sided 90% confidence intervals for measurements from the hand of each study subject. The half-
widths of the confidence intervals (a.k.a. the 90% margins of error on the means, or 90% MEMs) 
will depend heavily both on the number of subjects in a given skin-pigmentation group and 
on the intra-subject correlation among individual PA contrasts. Figure 5 shows how the 90% 
MEMs vary with intra-subject correlation for exemplar skin-pigmentation group sizes of 3, 5, and 
7 per group when the total sample size is 30 healthy volunteers. The 90% MEMs (in SD units) 
are for the group means of measurements taken from the hands of study subjects. They are 
calculated using the method of Donner under the assumptions (1) that PA contrasts will be 
determined on exactly 2 vessels per hand or exactly 3 vessels per hand, and (2)  that the 
denominator degrees of freedom will be 12 based on all three skin-pigmentation groups being 
represented among the 30 study subjects. Inasmuch as the intra-subject correlation is unlikely to 

 
33
be >0.5, the range of 90% margins of error shown in Figure D indicate that 30 healthy volunteers 
should provide sufficient precision to meet its research goals. For Groups 2-4, a sample size of 50 
melanoma subjects produces a symmetric two-sided 95% confidence interval on the mean 
difference between methods that has a half-width of 0.258 SD units, where one SD unit is one 
standard deviation of the difference between methods. In each group, the CTC detection rates via 
the in vivo Cytophone versus the in vitro methods will be compared in fifty samples from 
melanoma subjects via one-sided McNemar’s test at 5% alpha to determine whether in vivo 
diagnosis has a significantly increased detection rate. To calculate power of the planned test, we 
assume that, when the two methods produce a discordant pair of results on the same sample (i.e., 
when the in vivo Cytophone disagrees with the in vitro method on whether CTCs have been 
detected), the odds ratio is 10-fold in favor of in vivo data having detected the CTCs. Figure 6 
shows how power to detect the 10-fold odds ratio varies with the proportion of discordant pairs of 
assay results among a fixed sample size of N=50 melanoma subjects. Since the subjects with a 
current or recent history of melanoma are expected to have CTCs but have them at concentrations 
at or below the detection limits of existing in vitro assay methods, the proportion of discordant 
assay-result pairs among subjects should be quite high if in vivo Cytophone works as advertised. 
Figure 6 demonstrates that 50 subjects should provide sufficient power to meet the proposed 
research goals. 
 
 

 
34
Data Storage 
Individual information will be protected in all data resulting from this study. No personal 
information will be collected other than basic demographic descriptors. The Cytophone’s results 
will be analyzed and documented. All data (completed instrument tools and observation notes) 
will be kept confidential. Original data will be kept on a password protected computer, stored in a 
locked cabinet in the researcher's office and destroyed three years after completion of the research. 
 
 

 
35
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37
Figures 
 
 
 
Figure 1. Clinical The Cytophone (left) and PA probe position on the hand (right). 
 
 
Figure 2. The principle of CTC detection with the Cytophone platform. 
 

 
38
 
Figure 3. In vivo The Cytophone’s detection of CTCs in healthy volunteers and patients. A typical 
ultrasound image of a vein (top) in the dorsum of the hand and the representative examples of PA 
traces from a healthy volunteer and a patient with melanoma before and after signal processing. 
 
 
Figure 4. Optical schematic of PAFC -based The Cytophone device. 
 
 
 
 

 
39
 
Figure 5. 90% Margin of MEM versus intra-subject correlation for exemplar skin-pigmentation 
group sizes of 3, 5, and 7 per group when the total sample size is 30. The 90% MEMs (in SD 
units) are for the group means of measurements taken from the hands of study subjects. They are 
calculated using the method of Donner
 under the assumptions (1) that PA contrasts will be 
determined on exactly 2 vessels/hand (“O” symbols) or exactly 3 vessels/hand (“X” symbols) and 
(2) that the denominator degrees of freedom will be 12 based on all three skin-pigmentation 
groups being represented among the 30 study subjects. 
 
 
Figure 6. Power versus proportion of discordant pairs of assay results among N=30 total samples 
from melanoma subjects when the odds ratio among discordant pairs is 10-fold in favor of the 
Cytophone showing the positive detection result. Power is power of the one-sided McNemar’s 
test at 5% alpha.