Evaluation of Nano-Pulse Stimulation (NPS) in Patients With Seborrheic Keratosis
Phase: NA
Conditions: Lesion Skin, Seborrheic Keratosis
Sponsor: Pulse Biosciences, Inc.

1 
 
 
 
PROTOCOL 
 
 
 
 
NANOPULSE SEBORRHEIC KERATOSIS STUDY 
(NP-SK-002) 
 
 
 
March 17, 2017 
Rev. 4/17/17 
Rev. 4/28/17 
Rev. 7/14/17 
 
 
 
 
 
 
 
 
 
CONFIDENTIAL 
 
 
 
 
 
 
 
 
 

2 
 
 
 
BACKGROUND 
The Pulse TX is a medical device intended to clear the skin of benign, 
undesired skin lesions as an alternative to surgery and other more ablative 
methods for removing benign lesions. The system is designed to deliver a 
timed series of very low energy, high voltage (lesser than the voltage of static 
electricity) pulses of a time length between 100 and 750 nanoseconds 
(billionths)   of a second. The non-thermal effect on tissue takes place in a 
very shallow depth of skin directly below the sterile treatment tip. The device 
emits significantly less energy than existing laser, electro-surgery or electro-
cautery equipment. 
 
Extensive in vitro research demonstrates a stimulation of a programmed cell 
death called apoptosis in a treated area, which is one of the presumed 
mechanisms for observations of clinical and histologic changes t o  c e l l s  in 
multiple studies of in-vivo treated tissue. In animal studies with over 1000 rats 
and mice, nano- pulsed devices have been demonstrated to reduce or 
eliminate b e n i g n  tumors with a clinically acceptable margin of safety. 
A pilot clinical trial was conducted under IRB oversight in 2012 with a similar 
version of the Pulse TX device, with the findings published in Experimental 
Dermatology. (See Attachment A) A total of ten basal cell carcinomas on 
the skin of three Subjects were treated with a range of settings. Seven of 
the ten lesions were free of basal cells at the conclusion of the study, two 
partially resolved, and one recurred. No scars were visible at the healed 
sites of any of the successfully treated lesions. 
 
PRIOR CLINICAL INVESTIGATIONS 
Study Name: 
 
NanoPulse Human Tissue Study 
Study Number: 
 
NP-ABD-001 
Device Named:  
Pulse TX System 
PI Name:  
 
David Kaufman, MD 
Co PI: 
 
 
Michele Martinez, RN 
Study Dates: 
 
November 2016 to January 2017 
 
A total of seven subjects participated in this prospective, single site, non-
randomized study.  All of the participants were females representing several 
Fitzpatrick Skin Tone Classifications, and ranging from the low 40’s to middle 
60’s in age.   
 

3 
 
 
 
The study was comprised of two sequential segments. The first segment called 
for the serialized use of the test device in specific locations on intact abdominal 
skin predetermined to be resected during a standard abdominoplasty. The 
primary endpoint of the first segment of the study was to establish that the 
trauma to the tested skin would be minimal. The appearance of each intact 
tested tissue location was comparable to that anticipated following any 
minor surgical insult.  
 
Lidocaine was injected prior to use of the Pulse TX skin exposure.  Subjects 
reported little or no sensation during the treatment session. Subjects further 
reported little or no discomfort after the Lidocaine effect had diminished. There 
were no adverse events, no reports of bleeding, and no scarring noted by the 
investigator at the end of the 60 day study. 
 
The second segment of the study called for the histological analysis of 
tissue samples prepared from the abdominal section that were resected at 
the time of the abdominoplasty. Internal and outside expert histologists were 
consulted. This second segment did not impact the participating subject. 
Consequently, Pulse Biosciences, Inc. secured IRB oversight concentrated on 
the first segment of the study. It should be noted however that the histological 
analysis to date confirms that the Pulse TX was successful in achieving its 
intended effect.  See Attachment B for a Summary Histological Report. 
 
PROPOSED SEBORRHEIC KERATOSIS STUDY 
Study Name: 
Nanopulse Seborrheic Keratosis Study  
Study Number: 
NP-SK-002    
Device Named: Pulse TX System 
PI Name:  
David Kaufman, MD 
PI Name:  
James Newman, MD 
PI Name:  
TBD 
Study Dates: 
TBD   
 
PURPOSE AND SCOPE OF THE STUDY 
The primary purpose of the study is to evaluate the use of the Pulse TX System 
when used to remove Seborrheic Keratosis (SK) lesions from the non-facial 
areas of healthy adult subjects.  These benign skin lesions are frequently 
located on the limbs and/or trunk and not considered to be pre-cancerous. 
Patients routinely seek their removal for cosmetic/aesthetic reasons.  SK 
lesions are typically treated using cryogen or a scalpel, or dessication with a 

4 
 
heat-based energy technology such as a laser or electrosurgery.   SKs do not 
self-resolve without treatment.   
STUDY OBJECTIVES 
The specific objectives of the study are as follows: 
 
• Document the non-treated appearance of off-face SK’s 
• Evaluate the clearance of SK’s in off-face locations after removal using 
the Pulse TX System vs. pretreated appearance of the same lesion 
• Evaluate the clearance of the treated SK’s at various points in time over 
several weeks following the initial procedure, compared to the pre-treated 
SK’s  
 
PRIMARY ENDPOINT 
• The primary endpoint of the study is the degree of clearing of benign SK 
lesions as rated by the independent reviewer’s assessment of 
photographs of the lesions at the final study visit. 
 
o 
A 4-point scale (0, 1, 2, 3) will be used wherein each lesion will be 
characterized as “Clear”, “Mostly Clear“, “Partially Clear”, “Not Clear”.   
 
 
 
 
 
 
 
o See Attachment C for a protocol specific to the Independent Review.  
 
SECONDARY ENDPOINTS 
• A secondary endpoint of the study is the degree of clearing by the final 
visit as rated by the treating physician’s assessment.  
 
o The same 4-point scale will be used as that used for the Photographic 
Review.  
Photographic Review of SK Lesions 
Rating Scale 
Clear 
Mostly  
Clear 
Partially Clear 
Not  
Clear 
0 
1 
2 
3 
Investigator Assessment of SK Lesions 
Rating Scale 
Clear 
Mostly  
Clear 
Partially Clear 
Not  

5 
 
 
 
 
 
 
 
 
 
• An additional secondary endpoint of the study is subject satisfaction with 
the SK’s area at the final study visit. A 5-point scale (0, 1, 2, 3 4) will be 
used wherein the Subject will be asked to rate their level of satisfaction 
with each SK area as “Satisfied”, “Mostly Satisfied”, “Partially Satisfied”, 
“Dissatisfied” Highly Dissatisfied”. 
 
Patient Satisfaction for each SK area at day 106 
Satisfied 
Mostly 
Satisfied 
Partially 
Satisfied 
Dissatisfied 
Highly 
Dissatisfied 
(worse) 
0 
1 
2 
3 
 
4 
 
SUCCESS CRITERIA  
• 
Independent Reviewer Assessment of Photograph:  A score of “0” or 
“1” at the last follow-up will represent a successful outcome for that 
lesion.   The study will be considered a success if 65% of treated 
lesions are reduced by a score of 2 or more points on the lesion 
scale 
• 
Physician Assessment: A score of “0” or “1” at the last follow-up will 
represent a successful outcome for that lesion.  Success for this 
criteria is 65% of lesions rated as a 0 or 1 on the scale. 
• 
Patient Assessment: A score of “0” or “1” at the last follow-up will 
represent a successful outcome for that lesion.  Success for this 
criteria will be 65% of lesion areas evaluated as “Satisfied” or “Mostly 
Satisfied”. 
• 
The overall study will be considered a success if 2 of the 3 criteria 
listed above are met. 
 
Clear 
0 
1 
2 
3 

6 
 
STUDY DESIGN 
A prospective, randomized, open label, multi-site, NSR study design 
where each subject serves as his or her own control will be employed. A 
Principle Investigator will be identified at each of up to five participating sites. A 
total of up to 60 Subjects will be consented and enrolled with anticipated 
enrollment expected to be uniformly distributed across sites. The PI will 
consider the study inclusion and exclusion criteria and invite appropriate 
patients to participate in the study.  
No significant risk is posed by either the use of the Pulse TX System or 
the study process. While safety can never be taken lightly, this study is not 
meant to measure, monitor or analyze any significant disease or disorder for 
which medical treatment is mandatory. (See additional information regarding 
Non-Significant Risk statement in the following pages.) 
 
ANALYTICAL METHODS 
Three sets of data will be analyzed: 
1. Clinical observational data as derived from the examinations. 
2. Data captured during the photograph review process.  
3. Patient self-reported symptoms and observational data. 
 
The statistical analysis for this single-arm NSR study will be performed after 
collection of safety, effectiveness and secondary outcomes during the baseline 
period and each evaluation interval. 
  
• Analysis of primary endpoints in intent-to-treat population  
• Analysis of primary and secondary endpoints in the per protocol population  
• The calculation of the change from baseline in the treated lesion size and 
appearance will be adjusted by the observed effect on the untreated lesion 
change in size and appearance 
• Exploratory Data Analysis to describe distributions and to explore trending 
• Repeated measures ANOVA or non-parametric equivalent to compare 
means 
• Likelihood ratio statistics to assess categorical data 
• Chi-Square Tests to compare binary proportions 
• Exact probabilities will be computed for any inferential analysis performed 
• 95% confidence levels will be calculated using exact methods 
• Agreement statistics to estimate the inter- and intra-photographic reviewer 
variability  
• All analysis will be adjusted for the correlated nature of the data since more 
than one lesion is being sampled from the host subject 
 

7 
 
All tables and listings will be produced and stored in SAS datasets.  No data 
imputations will be performed. 
 
SAMPLE SIZE ESTIMATE 
A one group 2 test with a 0.050 two-sided significance level will have 91% 
power to detect the difference between the Null hypothesis proportion 0, of 
0.510 (51%) and the Alternative proportion, A, of 0.650 (65%) when the 
sample size is 135. 
DEVICE DESCRIPTION 
The Pulse TX System consists of an electrical pulse generator (similar to 
devices used to electro-coagulate tissue) combined with a hand-piece which 
is held by the clinician during application of pulses to the skin surface. The 
hand-piece is coupled to a sterile, single patient-use treatment tip. The tip has 
an array of pins no greater than 1.27 mm in diameter (18 gauge) which 
penetrate the skin between 1 and 3 mm. Power settings within a minimum 
and maximum range are available for selection by the physician, and are 
software limited to defined setting for each treatment tip by the sponsor. Once 
the electrical pulse generator is turned on and a power setting is selected, a 
foot switch triggers a sequence of pre-programmed electrical pulses to an 
area of skin directly beneath the treatment tip.  
 
A common commercially available sterile coupling gel is applied to the skin 
or treatment tip surface to ensure good electrical coupling to the tissue. 
 
The three system components are as follows: 
 
1. Pulse Generator including a built-in touch screen for setting selection, 
a power cord, and a mandatory foot pedal for activation of a pulsing 
sequence 
2. Hand-piece (re-usable) 
3. Single patient-use Tip (five different versions available, described 
below) 
 
Pulse TX Graphic 
 
 
LCD touch screen 
Handpiece holder 
 
 
 
 
 
 
 
    Hand piece holder 
 
handpiece receptacle 

8 
 
 
 
Footswitch connector 
 
Locking brakes 
 
 
Figure 1. Pulse generator 
Electrode Handpiece 
A reusable handpiece allows the operator to connect the single-use patient treatment tip 
to the generator. One end of the handpiece connects to the generator; the other end 
connects to the treatment tip. The handpiece can be cleaned and after each use. 
 
Single-use Treatment Tip 
A sterile single-use patient Treatment Tip attaches to the hand-piece. The tip delivers the 
nano pulses to the area to be treated through an array of micro-needles.  Five tip sizes 
are available: 
• 
2.5 mm x 2.5 mm Treatment tip 
• 
5 mm x 5 mm Treatment Tip 
• 
7.5 x 7.5 mm Treatment Tip 
• 
10 mm x 10 mm Treatment Tip 
 
 
 

9 
 

 
10 
SITE/INVESTIGATOR SELECTION  
Up to 5 study sites will be selected to participate in the study.  Two of the five sites 
have been identified.  The additional sites have not yet been determined. The table 
below provides additional information 
 
NAME OF STUDY SITE 
ADDRESS OF STUDY SITE 
Site 1: Aesthetic Artistry Surgical 
and Medical Center or AASMC 
2220 E Bidwell St  
Folsom, CA 95630 
T 916-983-9895 
David Kaufman, MD 
Michele Martinez, RN 
2220 E Bidwell St  
Folsom, CA 95630 
T 916-983-9895 
 
 
Site 2: Aesthetic Center  
James Newman, MD 
Melba Herrera, MD 
1795 El Camino Real 
Palo Alto, CA 94306 
T 650 321 3223 
 
Same Investigators at alternate 
location 
316 South Eldorado Street, Suite 105 
San Mateo, CA650-340-7200 
T 650 321 3223 
 
 
 
 
TBD 
 
NAME OF STUDY MONITOR 
 
Pamela M. Buckman, MSN  
Consultant , Pulse Biosciences 
2800 Pleasant Hill Rd., Suite 175 
Pleasant Hill, CA 94523 
T  925 980 7007 
F  925 705 7381 
pmbuckman@gmail.com 
 
Site #1 above will be the first to initiate the study.  This site and the Investigators have 
been approved by BioMed for a study that is currently open but not active. The required 
forms and information about subsequent sites and Investigators will be provided to 
BioMed prior to any study related activities at the site in questions. Prior to enrolling 
a Subject at any particular site the investigator and appropriate members of the study 
team at that site will participate in a comprehensive training program designed by 
Pulse. 
 

 
11 
SUBJECT RECRUITMENT AND SELECTION 
Convenience sampling will be used to enroll subjects exclusively from the patient 
population at each of the sites. The planned enrollment is set at up to 15 subjects 
at each site for a total of 60.  Recruitment will be conducted via direct communication 
between potential Subjects with each PI and/or the designated staff.    
 
The invitation to participate will be extended to those patients who are known or 
present with SK lesions that they individually choose to have removed. The Informed 
Consent discussion and signature process will be conducted by the PI and/or the 
designated staff. Once consented, each Subject will be assigned a unique Study 
Identification Number.  See Attachment D for statistical discussion and analysis of 
proposed Subject population.  
 
Recruitment Statement   
If recruitment is not adequate to meet the study requirements, a counter card or other 
media format may be used.  See Attachment D for a proposed recruitment statement. 
The study Inclusion and Exclusion criteria are noted below. 
 
Inclusion Criteria:    
• Willing to sign the informed consent 
• Males or females 
• At least 18 years of age but less than 65 years of age 
• Has a clinical diagnosis of stable, clinically typical seborrheic keratosis 
• Medically determined candidate for at least 4 off-face SK lesions 
• Must have at least four treatable SK’s and be willing to have Pulse TX 
treatments on any three of the four treatable SK’s. Treatable SK’s must:  
• 
be at least 1 mm in height, and no greater than 3 mm in height.   
• 
not have a dimension perpendicular to the longest dimension of 
greater than 7.5mm or longer in any dimension than 20mm.. 
• Willing to have three of the designated SK’s treated in a single treatment 
session 
• Willing to return to the PI’s office for five additional study visits at specified 
intervals over 106 days 
• Agrees to high resolution photos of both the treated SK lesions and the 
untreated SK lesion  
• No subject identity will be possible via the “lesion-only” photograph 
• Has no evidence of active infection in the designated tissue prior to treatment 

 
12 
• Is not allergic to Lidocaine or Lidocaine-like products 
• Not pregnant or lactating 
• In the investigators’ opinion, be in good general health and unlikely subject to 
risks from treatment 
 
Exclusion Criteria: 
• Implantable electronic devices. e.g., automatic defibrillator 
• Active infection or history of infection within 90 previous days in designated 
test area 
• Not willing or able to sign the Informed Consent 
• Non English speaking or reading 
• Is known to be immune-compromised 
• Known to be keloid producer 
• On blood thinning medications 
• Diseases, conditions, or situations wherein  PI judges  that the patient is 
not appropriate for participation in the study 
 
NON SIGNIFICANT RISK STATEMENT 
The proposed study fits the criteria for a non-significant designation for the 
following reasons: 
• 
Subjects will be recruited for the study strictly on a voluntary basis 
• 
Participation in the study takes approximately 1 hour for the first 
treatment visit and 30 minutes for each of the five post treatment 
evaluation visits, which includes photographs of both treated and control 
lesion(s)  
• 
Localized anesthesia will be used to control discomfort at the time of 
the test device application. 
• 
After the test device is deployed and the anesthesia is dissipated, there 
may be mild, localized discomfort at the site of the treatment. A small 
scab or crust may develop. This occurrence is transient and should 
resolve without intervention. 
• 
Transient and minor discomfort at the treated site and appearance of a 
small scab typically occurs when other standard treatments are used, i.e., 
cryogen to remove SK’s. Use of the Pulse TX does not raise any additional 
concerns compared to current methods of lesion removal 
• 
There is no loss of privacy as no study records will be viewed or retrieved 
by anyone other than the study team members. 

 
13 
• 
Protected information will not be captured 
• 
Subjects are protected by a coding system to de-identify them 
 
STUDY PROCEDURE 
The following is an example of the Subject related activities to be 
completed for each Subject in the study. The exact order may differ 
depending on Subject/Pl schedules and preferences. 
 
Activities prior to or on same day as study enrollment and occur on same 
day as treatment visit: 
• 
evaluation for Inclusion/Exclusion criteria 
• 
sign the consent form prior to any study activities 
• 
receive a copy of the signed consent form 
 
Activities on 1st Study Visit Day 
• 
Four SK’s are selected that meet the study criteria for treatable SK’s.  If 
more than four lesions are present, the patient can state a preference for 
which 4 lesions are included in the study 
• 
Photographs of each of the four selected SK’s will be taken prior to 
anesthesia or treatment 
• 
Local anesthesia w i l l  b e  applied to the four selected SK’s.   
• 
Using a randomization method, exactly three of the selected SK’s will be 
designated to be treated and one will be designated as an untreated 
control  
• 
The test device will be deployed to the selected SK's according to the 
randomized order 
• 
a light bandage will be applied 
• 
Subject will be discharged 
 
Activities on 2nd, 3rd, 4th, 5th and 6th Study Visit Days 
• 
Photographs of each of the 3 treated lesions and one non-treated lesion 
will be taken  
• 
Each lesion will be clinically assessed and the appearance will be rated 
according to specified criteria.  
• 
Any adverse events will be identified and documented. 
 
 

 
14 
RANGE OF DAYS FOR STUDY VISIT INTERVALS 
STUDY VISITS 
TARGET VISIT DATES 
VISIT DATE RANGES 
1 
Treatment Day 
NA 
2 
1 week post TX 
7 to 9 days post TX 
3 
1 month post TX 
28 to 35 days post TX 
4 
60 days post TX 
50 to 60 days post TX 
5 
90  post TX 
80 to 90 days post TX 
6 
106 days post 
100 to 111 days post 
 
SUBJECT COMPENSATION 
Each subject will be compensated for any inconvenience that study participation 
may represent. Specifically, each patient will make 6 total visits to the surgeon's 
office (approximately 4 hrs. total time).  In return for this time commitment, the patient 
will receive $200 for the treatment visit, $100 for each of the 5 follow up visits and $400 
bonus for completing all of the scheduled visits.  The total amount of compensation will 
be $1,100. 
 
STUDY BENEFITS 
A volunteer for the proposed study is someone who has elected removal of benign 
lesion(s).  Such lesions are typically removed via cryogen or a scalpel. Participation in 
the study offers another technique to accomplish the same thing as the typical tools.  
Experience with the Pulse TX to date offers some confidence that the lesions will be 
successfully removed although no assurances will be offered. The information learned 
may contribute to the ultimate use of a more safe and effective device available 
to ablate tissue in future patients. 
 
STUDY/DEVICE RISKS 
The Investigators for the study are experienced physicians. Use of the Pulse TX 
System poses very minimal risk, including scabbing, minor skin pigment changes or 
scarring and minor discomfort which can be further minimized by covering the spot 
with a small bandage. Typical wound complications such as infection, bleeding and 
discomfort which requires prescription medication for relief are not anticipated. If 
they should occur they would be considered an unanticipated adverse event and 
managed as such. If the Subject has any questions or concerns about their medical 
condition or if an unforeseen event should occur the subject will be advised to 
contact the PIs or their designate.    
 

 
15 
SITE TRAINING PLANS 
Training to be conducted can be divided into two pertinent categories. First is training 
on use of the Pulse TX System from a technical and clinical perspective. The PIs 
and their designated assistants will be instructed in aspects of set up, application, 
management and maintenance of the Pulse TX System. Those persons conducting 
the training will be Pulse Bioscience technical experts in this area of expertise. A 
select group of these experts will be present during the testing sessions to provide 
support as needed. Training will include a dry run. The second category is training 
in the clinical study requirements and processes. The study team members at the 
Study Site will be trained via the pertinent documents and files as well as the planned 
logistics to perform the study according to the protocol and applicable IRS, GCP and 
Pulse requirements. 
 
INSTITUTIONAL REVIEW BOARD OVERSIGHT 
The following Institutional Review Board will be asked to review and approve the 
proposed study: 
 
Biomedical Research Institute of America 
P.O. Box 600870 
San Diego, CA 92160  
T - 619 282 9997 
F - 619 282 9998 
 
If the Subject has any questions or concerns about the study process, they will be 
advised to contact Biomed at 619-282-9997 
 
 
 
STUDY MANAGEMENT AND MONITORING PLAN 
Pulse Biosciences, and/or their designated representatives are solely responsible for 
the proposed study.  Pulse will take the necessary steps to assure that the study is 
conducted in accordance with all regulating authorities as well as applicable 
Standard Operating Procedures. The Study Monitor will oversee the conduct of the 
study on a continual basis. Minimal Monitoring visits will be conducted at the 
following intervals: Site Qualification Visit, Study Initiation Visit, Interim Study Visit and 
Study Close-Out Visit. 
 
 
 

 
16 
 
ATTACHMENT A 
 
ARTICLE REPRINT 
 
NUCCITELLI 
ARTICLE.pdf
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
17 
 
ATTACHMENT B 
 
HISTOLOGY REPORT 
 
 

 
18 
A Summary Histology Report of a Dose Ranging Study of Nano-Pulsed Cell 
Stimulation in a Pre-Excised Human Tissue Model 
 
Microscopic samples of abdominal tissue from seven subjects treated with a novel non-
thermal energy device that delivers short low energy pulses to a controlled tissue 
volume have been reviewed in a detailed histologic analysis.  The treatment intervals 
of the targeted normal abdominal tissue ranged from 10 hours to 60 days prior to a 
previously scheduled abdominoplasty procedure in which the treated tissue was 
demarcated six months prior to excision.  Six treatment energy levels are reviewed per 
patient per time interval.   Various tissue staining techniques intended to identify 
specific cellular changes and tissue morphology were utilized in the histologic analysis 
to characterize the initial tissue responses and the subsequent recovery processes, 
and sequential findings for each of the six energy levels were compared to normal 
control punch biopsies in the same patient. 
 
Epidermal Changes:  Review of the changes of the entire thickness of the 
epidermal/dermal layer and subcutaneous fat from day 1 through day 60 was 
performed.  The primary changes due to the non-thermal energy exposure were 
observed in the treated skin within the epidermal layer of skin.  At day one post-
treatment, tissue samples from all subjects showed evidence of “ghost cells” in the 
epidermal layer characterized by intact cell membranes and absence of nuclei within 
those cells, which indicates a non-viability of those cells.  The non-viability of epidermal 
cells was full-thickness and complete for all energy settings by 1 day post-treatment.  
In many patients, hair follicles and eccrine glands within the dermal layer of the skin 
were also visible for histologic review.  In specimens in which hair follicles and eccrine 
ducts were visible, there is partial (50%) to full necrosis of the upper portions of the hair 
follicles and eccrine ducts.  5 days post-treatment epidermal changes range from 50% 
to 90% healing (50% - 90% viable epidermal cells).  Hair follicles and eccrine ducts 
when visible in the tissue samples show focal dyskeratotic cells within the hair follicles 
at 5 and 15 days consistent with partial healing at 5 days and complete recovery by 15 
days.  Eccrine ducts often showed focal squamous metaplasia, a sign of re-
epithelialization.  By 15 days the epidermis layer had returned to normal in almost all 
cases.  By 60 days the epidermis, hair follicles, and eccrine glands had all completely 
returned to normal.  Of note in two patients treated at the highest energy level, 
epidermal necrosis was followed by a formation of an inflammatory eschar.  This 
formation healed by 60 days with some epidermal flattening and minimal papillary 
dermal fibrosis. 
 
 
 

 
19 
Alterations of dermal collagen:  Alterations in the dermal collagen observed were 
minimal, with no evidence of thermal injury.  In several tissue samples exposed to 
treatment levels five and six, there was focal papillary dermal necrosis evident.  This 
effect was observed at the 1 day and 5 day intervals, but was not observed at the 15-
60 day intervals.  In two patients at treated at the highest energy level, there was some 
parallel fibrosis of the dermal collagen bundles at 60 days. 
 
Elastic tissue staining:  Elastic tissue remained intact in the vast majority of patients.  
In treatment levels 4, 5 and 6 there was occasional slight decrease in elastic fiber noted 
in earlier specimens, at days 1, 5 and 15, with return of elastic fibers observed by days 
30 and 60.  Taken in summary with the results of the trichrome collagen stain, these 
findings indicate relative minimal effects on the dermis. This predicts a very low risk of 
scarring. The risk is greatest in treatment level 6. 
 
Melanocytic density:  The number of melanocytes was observed using a MITF 
immunostain.  Staining for melanocytes shows a marked loss of melanocytes in the 
treatment areas at day one.  By day 15 the number of melanocytes return to normal 
density, comparable to the control specimens.  This was observed through 60 days.  
The rapid return of the melanocytic density to levels comparable to control should be 
consistent with a relative normalization of skin pigmentation over time 
 
Dermal fibroblasts:  The number of dermal fibroblasts appear to decrease at day 1 
and day 5.  However, by day 15, 30 and 60 the number of dermal fibroblasts was 
equivalent to pretreatment samples.  This suggests a temporary decrease in the 
number of dermal fibroblasts after treatment and possibly some loss of dermal 
fibroblasts secondary to the treatment.  However, fibroblasts are recruited from normal 
surrounding skin and peripheral circulation and the population of fibroblasts returned 
to levels similar to the control in the 15-60 day time period. This finding suggests a 
recovery of fibroblasts and the associated normal capacity to rebuild connective tissue. 
 
Apoptosis:  Measurement of epidermal Caspace staining was performed using 
immune-histochemical stains to active Caspace 3. This analysis shows no significant 
expression within the epidermis in the 1 and 5 day post-treatment time frames tested.  
These findings do not provide clear evidence of apoptotic cell death during the 
analyzed time frames.   Therefore, the precise mechanism for the observation of “ghost 
cells” and associated cell death was not identifiable by this method. 
 
 
 
 

 
20 
Inflammation: Overall, the degree of dermal inflammation was minimal compared to 
other thermal or physical methods of intentionally damaging surface epidermal tissue.  
There is a small amount of inflammation seen at day 1 and day 5, however the amount 
of inflammation appears to be sparse and perivascular.  Of note, there were several 
samples which showed focal perivascular inflammation with fibrin deposition 
suggestive of low grade vascular damage.  This can be seen at day 5 through day 60.  
This appeared to have no clinical correlation to degree of epidermal necrosis or impact 
on healing. 
 
Summary:  The novel method of using low energy nano-pulse cellular stimulation on 
skin and subcutaneous tissue at six different energy settings was observed to lead to 
a predictable loss of the epidermal layer of skin at all energy levels by one day 
posttreatment.  The lack of observed effect on dermal collagen suggest that the effect 
is non-thermal, with a relatively low level of inflammation given the amount of epidermal 
damage.    This lack of inflammatory effect is consistent with preservation of fibroblasts, 
elastin, and melanocyte recovery.  The transient effect on deeper cellular structures in 
the dermis suggest an affinity of nanopulse energy for highly cellular tissue, and a 
sparing effect on the less cellular connective tissue of the collagen layer. 
 
Author Information 
 
Darius Mehregan, MD 
Pinkus Dermatopathology Laboratory 
Hermann Pinkus, Chairman, Dept. of Dermatology 
Wayne State University 
1314 North Macomb St. 
Monroe, MI 48162 
T 734 242 6870 
F 734 242 4962 
 
 
 
 
 
 
 
 
 
 
 

 
21 
ATTACHMENT C 
 
INDEPENDENT PHOTOGRAPHIC REVIEW 
 
 
 
 
 
 

 
22 
INDEPENDENT PHOTOGRAPHIC REVIEW 
STUDY PROTOCOL 
BACKGROUND 
The NanoPulse System is a medical device intended to create a controlled non-
thermal reduction or clearing of targeted tissue areas as an alternative to excisional 
surgery and other more thermally ablative methods for removing undesired tissue 
or cells. The system deployed in the study is designed to deliver a timed series of 
very low energy, high voltage (similar to the voltage of static electricity) pulses of a 
time length between 100 and 750 nanoseconds (billionths) of a second. The non-
thermal effect takes place in a shallow depth of tissue directly below the sterile 
treatment tip. The device emits significantly less energy than other currently used 
electro-surgery or electro-cautery equipment.   
 
In several well-designed animal studies, nano- pulsed devices 
have been 
demonstrated to reduce or eliminate skin tumors with an acceptable margin of 
safety. An NSR pilot clinical trial was conducted under IRB oversight beginning in 
2012 with a similar version of the Nano Pulse device.  A total of ten basal cell 
carcinomas on the skin of three Subjects were treated with controlled energy 
settings with a varying number of pulses. Seven of the ten treated areas were free of 
basal cells at the conclusion of the study, two partially resolved, and one recurred. 
No scars were visible at the healed sites of any of the successfully treated basal 
cell carcinomas.  
 
A Human Tissue Study was conducted in late 2016 to early 2017 for the primary 
purpose of collecting biopsied samples of human tissue that had been tested with 
controlled exposures to the NanoPulse System at varying energy levels and number 
of pulses, and followed at 5 standardized time intervals up to 60 days.  This single site 
study enrolled 5 Subjects who were previously identified candidates for 
abdominoplasty.  Each Subject provided 30 treated areas to be biopsied. 
 
A  Photographic Review Study was performed including images of 150 non-thermal 
lesions that were created among the enrolled Subjects. For each of these 150 lesions, 
specific characteristics associated with normal wound healing were evaluated and 
rated according to a predetermined scale.  This Photographic Review Study was 
executed according to a comprehensive Protocol including Pre Training and followed 
by Intra- and Inter-rater analysis.  The described Independent Photographic Review 
Study serves as a model for the proposed Photographic Review SK Study. 
 
 
 

 
23 
PURPOSE of the PHOTOGRAPHIC REVIEW 
 
The independent review of the lesions photographed at each evaluation interval is 
being conducted to standardize the interpretation of the lesion clearance and 
appearance over time. The centralized independent review of the photographs can 
also reduce potential bias in these types of clinical trials where the assessment of the 
images can be subjective. Ensuring consistency and lack of bias is a critical issue for 
the Sponsor and FDA. The image acquisition and interpretation protocols help 
standardize the process across the clinical sites. A central review allows an auditable, 
rigorous, and uniform process of evaluation, thus  achieving higher confidence in the 
integrity of the data.  
 
OBJECTIVES and SUCCESS CRITERIA 
1st objective is to confirm that reported ratings are reproducible over time when 
considering one reviewer’s ratings at two different times so the agreement between the 
two time intervals should be at least 80% 
 
2nd objective is to confirm that reported ratings are reproducible when considering 
responses among 3 reviewer’s ratings when each is compared to the other two so the 
agreement between and among the reviews should be at least 80% 
 
REVIEWER SELECTION 
 Not affiliated with the study 
2-3 reviewers qualified by experience with evaluating skin lesions and wound 
healing 
Multiple locations  
Independent of each other 
 
PHOTOGRAPH SELECTION & SET UP 
• 
For each lesion in each of the 60 patients, five post-treatment photos of that 
lesion will be printed in a randomized order, along with an identified baseline 
photograph of that same lesion. Consistent standard of  photographic analysis 
to select all images to be reviewed at all time points according to uniform criteria 
• 
Images will be masked to time interval, energy settings, size of treatment 
tip,and specific subject identifiers. 
 
 
 
 
 

 
24 
STUDY PROCESS 
MATERIALS NEEDED 
1.  Set of photographs (Training/Reference Set) to be given to each of 
the reviewers 
• One set of images representing each SK lesion rating 
option and labeled with the rating it represents  
• One set of images representing  each of the skin 
characteristics to be rated 
• Narrative description of each of the rating options 
• No time intervals displayed 
• No energy settings displayed 
 
2.  Set of photographs (Scoring Set) to be given to each of the reviewers 
• Images are restricted to those taken at each of the visit 
intervals i.e., 7 days, 30 days, 60 days, 90  days and 106 
days post initial treatment 
• Images do not include those displayed in Training Set 
• Narrative description of each of the rating options 
• No time intervals displayed 
• No energy settings displayed 
 
3.  Set of photographs (V/V Set) to be given to each of the reviewers 
• Redistribute, rearrange images for at least 50% of the 
randomly chosen images  
• Renumber image identifiers 
 
REVIEWER TRAINING  
The training session may be done in person or via use of electronic 
communication and media formats.  Steps to be taken for the training 
session are as follows:  
• Send Training/Reference Set of images to reviewer 
• Set up telephone training session (options: Go to Meeting or similar).  
Should take about 15-30 minutes 
• Use consistent trainer for each reviewer session 
• Reviewer to keep Training/Reference Set of images to use as 
reference during the Review session 
• Trainer to document time, date, location, attendees, communication 
format and the topics discussed, etc. 

 
25 
REVIEWER SESSION 
The Review session may be done in person or via use of electronic 
communication and media formats.  Steps to be taken for the Review 
Sessions are as follows:  
• Send Scoring Set of images to reviewer 
• Set up telephone call (options: Go to Meeting or similar). Should take 
about 10 minutes to assure understanding of process 
• Use consistent contact person for each reviewer call 
• Set up deadline date for completion of the review 
• Contact person to document time, date, location, attendees, topics 
covered and communication format. 
• Instruct the Reviewer to return the Scored set of images but retain 
the Training/Reference set 
VALIDATION SESSION  
The Validation session may be done in person or via use of electronic 
communication and media formats.  Steps to be taken for the Validation 
Sessions are as follows:  
• Send V & V Set of images to Reviewers 
• Set up telephone call (options: Go to Meeting or similar). Should take 
about 10 minutes to assure understanding of process 
• Use consistent contact person for each reviewer call 
• Set up deadline date for completion of the review 
• Contact person to document time, date, location, attendees, topics 
covered, and communication format. 
• Instruct the Reviewer to return all study materials 
NOTE:  Inter- and intra-rater reliability tests are done to confirm that… 
1.  a reviewer can look at the same image at two different times 
and consistently rate the characteristic in both instances. (Intra-
rater Reliability) and 
2. reviewers are in agreement with each other when they rate the 
same characteristics under the same circumstances and 
looking at the same images (Inter-rater Reliability)  
DATA MANAGEMENT 
All data captured for the Independent Photographic Review Study will be entered into 
a database specifically designed for this study.  
 
 

 
26 
 
ATTACHMENT D 
 
RECRUITMENT STATEMENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
27 
 
RECRUITMENT STATEMENT 
 
 
Are you bothered by small dark spots on your limbs or torso? 
 
Are the spots raised? 
 
Are there at least 4 spots? 
 
Would you like to have them removed? 
 
If you are a good candidate to have the spots removed, you may be eligible to 
participate in a scientific study.   
 
This clinical study provides financial compensation for study-qualified 
participants.   
 
For more information about this study, please call our Patient Care Coordinator, 
xxxxxxx at xxxxxxxxx.    
 
Study participation is limited.