Cryoanalgesia to Treat Post-Amputation Phantom Limb Pain: A Multicenter, Randomized, Double-Masked, Placebo-Controlled, Definitive Human Subjects Clinical Trial
Phase: NA
Conditions: Post-Amputation Phantom Limb Pain
Sponsor: University of California, San Diego

 
 
 
 
 
 
 
 
Title:  Cryoanalgesia to Treat Post-Amputation Phantom Limb Pain: A 
Multicenter, Randomized, Double-Masked, Placebo-Controlled, 
Definitive Human Subjects Clinical Trial 
 
 
NCT number:  NCT03449667 
 
 
Document date:  January 5, 2022 
 

 
 
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UCSD Human Research Protections Program 
New Biomedical Application 
RESEARCH PLAN 
Instructions for completing the Research Plan are available on the HRPP website. 
The headings on this set of instructions correspond to the headings of the Research Plan. 
General Instructions: Enter a response for all topic headings.  
Enter “Not Applicable” rather than leaving an item blank if the item does not apply to this project. 
Version date: 9/30/2013 
1. 
PROJECT TITLE 
Cryoanalgesia to Treat Post-Amputation Phantom Limb Pain: A Multicenter, Randomized, 
Double-Masked, Placebo-Controlled, Definitive Human Subjects Clinical Trial 
 
 
2. 
PRINCIPAL INVESTIGATOR  
Brian M. Ilfeld, MD, MS 
 
3. 
FACILITIES 
UCSD hospitals and the UCSD CTRI 
 
4. 
ESTIMATED DURATION OF THE STUDY 
Five years (1 year regulatory and preparation, 2 years enrollment, 1 year follow-up, 1 year analysis 
and publication) 
 
5. 
LAY LANGUAGE SUMMARY OR SYNOPSIS (no more than one paragraph) 
When a limb is severed, pain perceived in the part of the body that no longer exists often develops 
and is called “phantom limb” pain.  Unfortunately, phantom pain goes away in only 16% of afflicted 
individuals, and there is currently no reliable definitive treatment. The exact reason that phantom limb 
pain occurs is unclear, but when a nerve is cut—as happens with an amputation—changes occur in 
the brain and spinal cord that actually increase with worsening phantom pain.  These abnormal 
changes may often be corrected by putting local anesthetic—called a “nerve block”—on the injured 
nerve, effectively keeping any “bad signals” from reaching the brain with a simultaneous resolution of 
the phantom limb pain.  However, when the nerve block resolves after a few hours, the phantom pain 
returns.  But, this demonstrates that the brain abnormalities—and phantom pain—that occur with an 
amputation are not necessarily fixed, and may be dependent upon the “bad” signals being sent from 
the injured nerve(s), suggesting that a very long peripheral nerve block—lasting many months rather 
than hours—may permanently reverse the abnormal changes in the brain, and provide definitive 
relief from phantom pain.  A prolonged nerve block lasting a few months may be provided by freezing 
the nerve using a process called “cryoneurolysis”.  The ultimate objective of the proposed 
research study is to determine if cryoanalgesia is an effective treatment for intractable post-
amputation phantom limb pain.  The proposed research study will include subjects with an existing 
lower extremity amputation who experience intractable daily phantom limb pain.  A single ultrasound-
guided treatment of cryoneurolysis (or sham block—determined randomly like a flip of a coin) will be 
applied to the target nerve(s) involved with the phantom pain. Although not required, each subject 
may return four months later for the alternative treatment (if the first treatment is sham, then the 
second treatment would be cryoneurolysis) so that all participants have the option of receiving the 
active treatment.  Subjects will be followed for a total of 12 months with data collected by telephone. 
 
6. 
SPECIFIC AIMS 
The ultimate objective of the proposed research is to determine if cryoanalgesia is an effective 

 
 
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treatment for intractable post-amputation phantom limb pain. 
 
Primary Specific Aim:  To test the influence of a cryoanalgesia treatment as compared to 
sham/placebo on the intensity of existing, intractable phantom limb pain resulting from an 
amputation. 
 
Hypothesis 1:  Phantom limb pain intensity will be significantly decreased 4 months following 
one cryoneurolysis procedure (as measured by the Numeric Rating Scale within the Brief 
Pain Inventory). 
 
Secondary Specific Aim:  To test the influence of a cryoanalgesia treatment as compared to 
sham/placebo on the quality of life for individuals with intractable phantom limb pain 
resulting from an amputation. 
 
Hypothesis 2a:  Perception of well-being will be significantly improved 4 months following one 
cryoneurolysis procedure  (as measured with the Patient Global Impression of Change 
Scale). 
 
Hypothesis 2b:  Physical and emotional functioning will be significantly improved 4 months 
following one cryoneurolysis procedure (as measured with the Brief Pain Inventory). 
 
Hypothesis 2c:  Depression will be significantly decreased 4 months following one 
cryoneurolysis procedure (as measured with the Beck Depression Inventory). 
 
7. 
BACKGROUND AND SIGNIFICANCE 
Of American veteran and civilian amputees, 35-98% (depending on the study) develop chronic, 
intractable pain perceived as being from the missing limb, a phenomenon termed “phantom limb 
pain”.1-3  The pain is usually described as “shooting, stabbing, boring, squeezing, throbbing, and 
burning”.1,3  Unfortunately, phantom pain resolves in only 16% of afflicted individuals (with or without 
treatment).4  The rest will experience phantom pain for the remainder of their lives, with most 
becoming dependent upon chronic opioid use to gain even a small degree of relief.  There is 
currently no reliable, definitive treatment for phantom limb pain.5 
 
Current evidence suggests that when a nerve is severed, the barrage of nociceptive input triggers a 
complex interaction between the peripheral and central nervous system. Both systems are dynamic, 
and injury to peripheral nerves provokes changes in the dorsal horn, thalamus, and cerebral cortex 
which are referred to as “neuronal plasticity”.6  Reorganization at the level of the spinal cord may 
result in “sensitization” in which dorsal root ganglion cells become hyperactive, resulting in stump 
allodynia and hyperalgesia.7  However, it is the somatosensory cortex within the brain that creates a 
“map” of the body—each location represented in a specific area of the cortex (i.e. homunculus); and 
deafferentation of neural pathways often results in changes of the cortical somatotopic map.8  For 
example, the zone of the cortex representing the fingers may be invaded by adjacent areas following 
a hand amputation and subsequent deafferentation.9 
 
Imaging techniques such as functional MRI have documented a correlation between phantom limb 
pain and cortical reorganization—the more intense the phantom pain, the greater the cortical 
changes.8  When the neural input from an amputated limb was blocked with a single injection of local 
anesthetic (a peripheral nerve block) in 6 subjects, 3 had immediate, complete resolution of their 
phantom pain; and, within minutes the cortical abnormalities were corrected for these three 
individuals.10  Unfortunately, when the single-injection nerve block resolved after a few hours, the 
phantom pain returned.  But, this intriguing result suggests that a prolonged peripheral nerve 
block—lasting multiple weeks rather than hours—may permanently reorganize cortical pain 

 
 
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mapping, thus providing lasting relief from phantom pain. 
 
A “continuous peripheral nerve block”—administration of local anesthetic through a percutaneously-
inserted perineural catheter—may provide a prolonged block.11  In an uncontrolled series of 19 
patients with phantom pain treated with continuous blocks, pain intensity was halved at 1 and 6 
months.12  However, the sensory block using this technique is frequently incomplete and/or 
inconsistent; the infusion duration usually limited to less than one week due to the risk of infection 
and difficulty of carrying a large bag of local anesthetic; and catheter dislodgement is relatively 
common.11  A more reliable, complete block of longer duration would theoretically increase any 
treatment effects for phantom limb pain, while reducing these inconveniences and complications. 
 
An alternative analgesic technique is cryoneurolysis, 
consisting of the application of exceptionally low 
temperatures to reversibly ablate peripheral nerves, resulting 
in temporary pain relief termed “cryoanalgesia”.13  The 
intense cold temperature at the probe tip produces Wallerian 
degeneration—a reversible breakdown of the nerve axon—
subsequently inhibiting transmission of afferent and efferent 
signals.  Because the nerve endoneurium, perineurium, and 
epineurium remain intact, the axon regenerates along the 
exoskeleton at a rate of approximately 1-2 mm/day.14  While 
cryoneurolysis of peripheral nerves through surgical 
incisions has been commonly used to treat pain since 
1961,15 the development of cryo probes that may be inserted 
percutaneously promise a revolution in the use of this modality.  The combination of newly-designed 
cryoneurolysis devices (above right), the narrow-gauge probes (immediate right), and ultrasound16,17 
now make percutaneous cryoanalgesia as simple as placing a peripheral nerve block:  the probe tip 
is inserted adjacent to the target nerve under ultrasound guidance, and a series of 1-minute freezing 
cycles are administered followed by probe withdrawal.13  The procedure is essentially the same as 
placing an ultrasound-guided peripheral nerve block; however, instead of injecting local 
anesthetic, a gas circulates through the probe, inducing cold at the tip and freezing the target 
nerve.  Nothing remains within the patient and there is no external equipment to prepare or manage.  
Importantly, cryoneurolysis and the cryo probes are already approved by the United States Food and 
Drug Administration for the treatment of pain, including phantom limb pain, so no additional 
regulatory approval is required for the proposed clinical trial. 
 
Theoretical benefits of cryoneurolysis include an ultra-long duration of action without opioid 
involvement, no catheter management/removal, the lack of an infusion pump and anesthetic reservoir 
to carry, an extraordinarily-low risk of infection (approaching zero), and no risk of local anesthetic 
toxicity, catheter dislodgement or leakage.  With a single 8-minute percutaneous cryoneurolysis 
procedure consisting of several freeze/defrost cycles, an absolute truncation of nerve conduction is 
induced for 6-8 weeks with the complete restoration of nerve structure and function following 
remyelination.16-18 
 
Over 200,000 traumatic and surgical amputations occur annually within the United States alone;19-29 
with an estimated 1.6-million people living with an amputation, and this number is expected to double 
by 2050.30  A disproportionately large percentage of this population is comprised of active duty 
personnel and veterans due to combat trauma, peripheral vascular disease, and diabetes.31,32  The 
combination of increased munitions force,33 use of improvised explosive devices,26,32 and casualty 
survival rates has resulted in a dramatic increase in the percentage of recently-injured combat 

 
 
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veterans living with a traumatic amputation.23,27  Additionally, traumatic amputations have occurred in 
every major military conflict, leaving tens-of-thousands of United States Armed Forces veterans with 
missing limbs.  Furthermore, veterans undergo amputation due to peripheral vascular disease and 
diabetes at a rate 250% higher than the general population; over 10% of all male amputees within the 
United States each year are veterans.31 
 
Of veteran amputees, 35-98% (depending on the study) develop chronic, intractable phantom 
limb pain.1 Similarly, within the civilian population, the published incidence of phantom pain from 16 
different studies ranged from 50-95%.2,3,34   Phantom pain resolves in only 16% of afflicted 
individuals.4  Chronic pain greatly decreases quality-of-life and the chances of return to duty or 
civilian work;4,35 and the economic toll for chronic nonmalignant pain—including phantom pain—is 
over $100-billion annually within the United States29 
 
There is currently no reliable treatment for phantom limb pain.5  While more than 43 methods for 
treating phantom pain have been described,36 the placebo effect is common,37 and prolonged relief is 
experienced by fewer than 10% of treated patients (6% of untreated patients ultimately experience 
spontaneous resolution).38 Evidence of the intractable nature of phantom pain may be found in a 
survey of more than 10,000 amputees which reported a 1% treatment success rate.39  Therefore, 
the overwhelming majority of people suffering from phantom limb pain are dependent upon 
opioid analgesics to gain even a small degree of relief.  There are few data from randomized 
trials to guide treatment, leading the authors of a major review to conclude that there remains a 
substantial “gap between research and practice in the area of phantom limb pain”.5 
 
Given chronic pain’s enormous costs to individuals and society—and the intractable, currently-
untreatable nature of phantom limb pain with concurrent opioid dependence—it is imperative that an 
effective treatment be developed.  If our study demonstrates that ultrasound-guided percutaneous 
cryoneurolysis is a successful treatment, the resulting impact on the hundreds-of-thousands of 
Americans suffering from intractable phantom limb pain will be immediate and profound, as (1) 
healthcare providers within the United States Armed Forces, Veterans Affairs Medical Centers, and 
civilian hospitals already have expertise placing ultrasound-guided peripheral nerve blocks; (2) 
cryoneurolysis is a relatively inexpensive, single-procedure, outpatient treatment with few 
complications; and (3) cryoanalgesia is already approved by the United States Food and Drug 
Administration. 
 
8. 
PROGRESS REPORT 
We have completed a pilot study suggesting that a single percutaneous cryoneurolysis treatment 
holds great promise to provide very long-term—possibly permanent—phantom limb pain relief.  In the 
residual limb of five subjects, a local anesthetic-based peripheral nerve block was applied to the 
nerve corresponding to the location of the phantom pain (e.g., the sciatic nerve for phantom foot 
pain), with percutaneous cryoneurolysis subsequently administered in the same location.  Follow-up 
occurred after 3 and 6 months, and then every 6 months thereafter.  For the 3 subjects whose 
phantom pain resolved following local anesthetic injection (labeled “full responders” as defined with a 
numeric rating pain score [NRS] ≤ 1 following treatment), all three reported an NRS ≤ 1 for the 2-5 
year follow-up period; and, achieved this level of comfort without requiring any opioids, 
gabapentin, or additional analgesics (Figure below).  These subjects had upper or lower 
amputations due to peripheral vascular disease or trauma.  The remaining two subjects similarly 
experienced a significant decrease in their phantom limb pain immediately following cryoneurolysis of 
at least 2 points on the NRS (29% and 57% decreases), but did not have near-resolution of their 
phantom pain with the previous local anesthetic injections.  These two subjects continued to 

 
 
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experience decreased phantom pain for 5 months until both expired (cause of death unrelated to 
phantom pain treatment).  There were no cryoanalgesia-related complications.  Importantly, nearly 
identical results were reported by Prologo, and colleagues, in a similar pilot study involving 
21 subjects (currently In Press).  
 
Figure.  Pilot study results:  phantom limb pain measured on a Numeric Rating Scale (NRS) of 0-10 
prior to and following cryoneurolysis. “Partial responders” experienced decreased phantom pain 
following a local-anesthetic based peripheral nerve block on the NRS, but the NRS did not ultimately 
fall to ≤ 1. 
 
 
9. 
RESEARCH DESIGN AND METHODS 
This will be a multicenter, randomized, triple-masked (investigators, subjects, statisticians), 
sham/placebo-controlled, parallel (with optional crossover), human-subjects clinical trial to determine 
if cryoanalgesia is an effective treatment for intractable post-amputation phantom limb pain.  We 
have included a diverse group of recruitment sites that will provide a broad representative patients 
sample.  Study participants will be recruited at 7 centers, including 2 U.S. military, 2 Veterans Affairs, 
and 3 civilian university medical centers (1 private, 2 public), within a wide geographic range 
including the South, Midwest, East and West Coasts, providing a study sample with ethnic, racial, 
geographic and socioeconomic diversity. 
 
U.S. military medical centers: 
• Walter Reed National Military Medical Center, Bethesda, Maryland 
• Naval Medical Center San Diego, San Diego, California 
 
Veterans Affairs medical centers: 
• Palo Alto Veterans Affairs Medical Center, Palo Alto, California 
• Pittsburgh Veterans Affairs Healthcare System, Pittsburgh, Pennsylvania 
 
Civilian university medical centers: 
• Cleveland Clinic, Cleveland, Ohio 
• University of California San Diego, San Diego, California 
0 
2 
4 
6 
8 
10 
Months 
Pain Score  (NRS)  
  Pa
  Pa
  Re
  Re
  Re
0 
2 
4 
6 
8 
10 
Months 
Pain Score  (NRS)  
0 
2 
4 
6 
8 
10 
Months 
Pain Score  (NRS)  

 
 
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• University of Florida, Gainesville, Florida 
 
All protocols and study materials will be approved by each center’s Institutional Review Board; and, 
the study will be prospectively registered on the clinicaltrial.gov website.  The study will be overseen 
by both a medical monitor (Salim Hayek, MD, PhD; Case Western Reserve University; Cleveland, 
Ohio)—in essence a study subject advocate—as well as a Data Safety Monitoring Board comprised 
of the medical monitor, a physician familiar with the ethical conduct of clinical research, and 
statistician.  The Medical Monitor is responsible to oversee the safety of the research and report 
observations/findings to the IRB or a designated institutional official. The Research Monitor will 
review all unanticipated problems involving risks to subjects or others associated with the protocol 
and provide an independent report of the event to the IRB. The Research Monitor may discuss the 
research protocol with the investigators; shall have authority to stop a research protocol in progress, 
remove individual human subjects from a research protocol, and take whatever steps are necessary 
to protect the safety and well-being of human subjects until the IRB can assess the monitor's report; 
and shall have the responsibility to promptly report their observations and findings to the IRB or other 
designated official and the Human Research Protections Office.  The medical monitor and DSMB will 
review enrollment, study data, protocol violations, adverse events, and oversee all aspects of the 
clinical trial every one and six months, respectively, through data analysis.  All procedures are 
considered experimental given these are volunteers and they would not receive these treatments 
without study participation—while cleared by the FDA for use in all chronic and acute pain states, 
cryoneurolysis has not been demonstrated as effective to treat phantom limb pain, and it is not 
considered “standard-of-care” at either UCSD or other institutions. 
 
Enrollment.  Enrolling centers will recruit patients from four sources:  (1) surgical and chronic pain 
databases; (2) amputation, surgical, and chronic pain clinic referrals; (3) print and internet/web 
advertisements; and (4) clinicaltrials.gov.  Patients who are interested in the study will be required to 
give permission for a research coordinator or investigator to contact them to adhere to Health 
Insurance Portability and Accountability Act (HIPAA) requirements.  In addition, with IRB-approval, 
patients within multiple database types will be informed of the study via the United States postal 
service in the form of an IRB-approved letter.  The letters will include research contact information for 
patients with interest in study participation. 
 
Research coordinators or investigators will both explain the study protocol to interested patients, and 
subsequently review the inclusion/exclusion criteria.  Subjects meeting inclusion/exclusion criteria 
and desiring study participation will be scheduled for a diagnostic injection and cryoneurolysis 
procedure at the nearest enrolling center.  Written, informed consent will be obtained from each 
participant prior to any measurements and/or procedures.  It is anticipated that this will require 15-30 
minutes for each subject. 
 
The method of documenting consent will be using written informed consent forms approved by the 
local Institutional Review Board.  Subjects will be asked to make no changes to their analgesic 
regimen for at least 1 month prior to the cryoneurolysis procedure and continuing for 4 months until 
the measurement of the primary end point—for the duration of the study, all patients will be allowed 
to continue their pre-intervention analgesics.  In other words, subjects will continue taking the same 
analgesics during the study period as they were receiving prior to the study period, including their 
standard rescue analgesics—the study protocol simply freezes the analgesic regimen from 1 month 
prior to the intervention until the primary end point is measured 4 months later. 
 
Subjects will be asked to not eat or drink after midnight the night before the procedure.  For women 

 
 
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of childbearing age with the possibility of pregnancy, a sample of urine will be collected before any 
study interventions to confirm a non-pregnant state.  All subjects will have a peripheral intravenous 
(IV) catheter inserted, standard noninvasive monitors applied (blood pressure cuff, pulse oximeter, 5-
lead ECG), and oxygen administered via a facemask or nasal cannula.  Oral and intravenous 
sedatives and analgesics such as Midazolam, valium and fentanyl will be titrated for patient comfort if 
necessary, while ensuring that patients remained responsive to verbal cues.   
 
The specific nerves targeted will be the sciatic and femoral (or their distal branches).  The potential 
cryoneurolysis sites will be cleansed with chlorhexidine gluconate and isopropyl alcohol.  Using the 
optimal ultrasound transducer for the specific anatomic location and subject anatomy (linear vs 
curvilinear array), the target nerves will be identified in a transverse cross-sectional (short axis) view.  
A local anesthetic skin wheal will be raised adjacent to the ultrasound transducer and a Tuohy-tip 
needle will be inserted through the skin wheal in-plane beneath the ultrasound transducer and 
directed until the needle tip is immediately adjacent to the target nerve.  Local anesthetic (1-3 mL, 
lidocaine 2%) will be injected in divided doses with frequent aspiration.  This will be repeated for the 
second target nerve (either femoral or sciatic).  Within 20 minutes of the second injection, the 
subject’s limb pain level will be evaluated on the 0-10 NRS and if higher than at baseline prior to 
injection, the subject will NOT continue with treatment and their participation in the study will 
terminate upon discharge. 
 
Treatment group assignment (randomization).  Remaining subjects will be allocated to one of two 
possible treatments: 
 
 
 
1.  cryoneurolysis 
2.  sham cryoneurolysis (placebo control) 
 
Randomization will be stratified by enrolling institution in randomly chosen block sizes.  
Randomization lists will be created using computer-generated tables by the Cleveland Clinic.  
Treatment group assignment will be conveyed to the enrolling sites via the same secure web-based 
system (RedCap) used to collect and collate all post-intervention endpoints (see “Data Collection” 
paragraph below).  The first digit of the randomization numbers will be a letter denoting the enrolling 
center (of 7 possible); and, the next two digits will be integers beginning from 1 and increasing for 
each center.  Cryoneurolysis probes are available that either (1) pass nitrous oxide to the tip inducing 
freezing temperatures; or, (2) vent the nitrous oxide at the base of the probe so that no gas reaches 
the probe tip, resulting in no temperature change (PainBlocker, Epimed, Farmers Branch, Texas).  
Importantly, these probes are indistinguishable in appearance, and therefore treating physicians, 
subjects, and all clinical staff will be masked to treatment group assignment [only one individual at 
each institution will be unmasked and will provide the treating physician/investigator with the correct 
probe after opening the randomization envelope].  Unmasking will not occur until statistical analysis is 
complete (termed “triple masked”). 
 
Intervention.  The potential cryoneurolysis sites will be again cleansed with chlorhexidine gluconate 
and isopropyl alcohol.  With the same ultrasound transducer used to previously administer local 
anesthetic, the target nerve will again be identified in a transverse cross-sectional (short axis) view at 
or distal to the deposition of local anesthetic.  A cryoneurolysis device (PainBlocker, Epimed, 
Farmers Branch, Texas) will be used with the appropriate randomization-designated probe (either 
active or sham/placebo) and nitrous oxide (Figured in the Background section).  An angiocatheter 
will be inserted through a local anesthetic skin wheal in-plane beneath the ultrasound transducer and 
directed until the probe tip is immediately adjacent to the target nerve (lidocaine 2% will be 
administered, as needed, to anesthetize the angiocatheter track).  The angiocatheter needle will be 

 
 
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removed, leaving the angiocatheter through which the Epimed probe will be inserted until it is 
adjacent to the target nerve.  The cryoneurolysis device will be triggered using 3 cycles of 2-minute 
gas activation (active or sham) separated by 1-minute defrost periods.  For active probes, the nitrous 
oxide will be deployed to the tip where a drop in temperature to -70°C will result in cryoneurolysis.  
For the sham probes, the nitrous oxide will be vented prior to reaching the probe shaft, resulting in a 
lack of perineural temperature change.  The process will be repeated with the same treatment probe 
for the femoral nerve (e.g., both nerves will receive either active cryoneurolysis or sham/placebo, and 
not a mix of the two possible treatments). 
 
Prior to discharge, subjects will be provided with verbal and written instructions, the telephone and 
pager numbers of an investigator, and a copy of the Institutional Review Board-approved consent 
form.  Subjects will be provided with crutches if they desire, although we have found that nearly all 
patients treated with cryoneurolysis continue to ambulate using their prosthesis without difficulty.  
Subjects will be telephoned to answer any questions they may have the days following the 
intervention (Days 1-7, as desired by the subject and/or medically indicated).  The approximate 
duration of this visit will total 2-4 hours, from the time the subject enters the treatment facility until the 
time the depart. 
 
Optional crossover treatment.  Four to six months following the initial treatment, subjects may 
return for an optional repeated intervention procedure (“crossover”) with the alternative treatment 
(either active cryoneurolysis or sham/placebo), again in a double-masked fashion using the same 
protocol as described for the initial intervention.  The crossover treatment is not required for 
study participation, as the primary analyses will include a parallel study design for the initial 
intervention evaluated prior to any crossover treatment.  However, an optional crossover 
treatment will be offered to subjects for two reasons:  (1) it will ensure that all subjects have access to 
the proposed treatment, regardless of the treatment they are initially randomized to; and, (2) it will 
permit intra-subject differences between treatments to be analyzed (e.g., assessing treatment-effect 
heterogeneity, or the variability of the causal effect across individuals, which will would not be 
available from the parallel-group portion of the study alone).  These intra-subject differences will be 
secondary analyses, as there may be patient-selection bias regarding which subjects decide to have 
the crossover treatment (e.g., if the intervention is successful at greatly reducing phantom limb pain, 
then subjects receiving active cryoneurolysis during their initial treatment will be more likely to forgo 
the crossover treatment).  This crossover will not affect the primary analyses, which will involve a 
parallel group study design and investigate the effects of cryoneurolysis within 4 months of the initial 
intervention.  The approximate duration of a crossover visit will total 2-4 hours, from the time the 
subject enters the treatment facility until the time the depart. 
 
Following study completion, the results will be mailed to all enrolled subjects in written form using non-
technical (i.e. “layperson”) language. 
 
Outcome measurements (endpoints).  We have selected outcome measures that have established 
reliability and validity, with minimal inter-rater discordance, and are recommended for chronic pain 
clinical trials by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials 
(IMMPACT) consensus statement. The primary end point will be the difference in average daily 
phantom pain intensity at baseline and 4 months following the initial intervention (measured 
with the NRS as part of the Brief Pain Inventory). The primary analyses will compare the two 
treatments (inter-subject comparisons) during the initial treatment period in which half of the subjects 
will receive active cryoneurolysis and the other half a sham/placebo treatment.  Endpoints will be 
evaluated at baseline and post-treatment (Day 0), Days 1 and 7; and Months 1, 2, 3, 4, and 12, 

 
 
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(Table 1).  These same time points through Month 4 will be evaluated following the optional second 
(crossover) treatment. 
 
The questionnaire will differentiate among multiple dimensions of limb pain: 
 
Residual limb (“stump”) pain:  painful sensations localized to the portion of limb still present.  
Phantom limb sensations: non-painful sensations referred to the lost body part.  
Phantom limb pain:  
painful sensations referred to the lost body part.  
 
Each type of pain/sensation will be defined for subjects immediately prior to questionnaire application 
at each time point, and subjects will be instructed to specifically address phantom limb pain when 
responding to the various questions.  In addition, since there is a strong correlation between phantom 
and residual limb pain, we will specifically inquire about both types of pain.  Each time the 
questionnaire is applied, subjects will be instructed to respond for the previous 3 days (e.g., worst 
pain during the previous 3 days) because studies have suggested that patients have “increasing 
difficulty actually remembering symptom levels beyond the past several days.”  Exceptions will be on 
Day 1 for both the initial and crossover treatments because at these time points, the interest is in 
subjects’ experiences subsequent to the treatment and not prior to the intervention.  During these two 
days, subjects will be instructed to respond for the period of time since their treatment. 
 
Table 1.  Summary of post-enrollment assessments. 
Subjects: 
All 
Participating in Crossover 
* 
All 
Time Point Following: 
Initial Treatment 
Crossover Treatment 
Initial 
Time Point (Days): 
0 
1 
7 
 
 
 
 
0 
1 
7 
 
 
 
 
 
Time Point (Months): 
 
 
 
1 
2 
3 
4 
 
 
 
1 
2 
3 
4 
12 
Brief Pain Inventory (for 
Phantom Limb Pain) 
              
 
Residual Limb Pain (NRS) 
              
 
Patient Global Impression of 
Change Scale 
              
 
Beck Depression Inventory 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Non-Painful Phantom 
Sensations 
              
 
Phantom Limb Pain 
              
 
Residual Limb Pain 
              
 
* The second—or “crossover”—treatment intervention will occur between 4-6 months following the 
initial treatment 
 
 
Demographic and amputation history.  Subjects will have demographic data collected, including 

 
 
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age, sex, height, weight, educational level, employment status, marital status, current analgesic 
regimen (including adjuvants such as acupuncture), and U.S. military service (e.g., none, discharged, 
active).  In addition, amputation-specific data will include date of initial amputation, date(s) of 
subsequent surgical procedures, amputation etiology, amputation level, other amputations (with 
dates/etiology/pain), and prosthesis use. 
 
 
 
 
Hypothesis 1:  Phantom limb pain intensity will be significantly decreased 4 months following 
one cryoneurolysis procedure (as measured by the Numeric Rating Scale within the Brief 
Pain Inventory). 
 
 
Pain intensity.  Current/present, worst, least, and average phantom pain will be assessed using a 
Numeric Rating Scale (NRS) as part of the Brief Pain Inventory (short form), with the “average” pain 
score designated as the primary endpoint.  In addition, average and worst residual limb pain NRS will 
be recorded separately from the phantom pain scores.  The NRS is a highly-sensitive measure of 
pain intensity with numbers ranging from 0 to 10, zero equivalent to no pain and 10 equivalent to the 
worst imaginable pain.  The NRS has been demonstrated to be a valid and reliable measure in 
multiple pain states—including painful peripheral neuropathy specifically—and following analgesic 
interventions.  In addition, NRS scores correlate well with other measures of pain intensity, and 
demonstrate high test-retest reliability in chronic nociceptive and neuropathic pain states.  These 
NRS characteristics led to recent IMMPACT consensus recommendations for use of the 10-point 
NRS of pain intensity for chronic pain trials.  
 
 
 
 
Hypothesis 2a:  Perception of well-being will be significantly improved 4 months following one 
cryoneurolysis procedure  (as measured with the Patient Global Impression of Change 
Scale). 
 
 
 
 
Health-related quality of life.  While single-item measures of pain level/relief are currently the most 
reliable and valid options to measure pain intensity, the multidimensional aspect of the pain 
experience has led consensus recommendations for use of “global” measures of improvement in 
chronic pain trials. The Patient Global Impression of Change Scale is one such measure allowing 
patient evaluation of integrated treatment effects.  This measure is a 7-point ordinal scale requiring 
the subject to rate the current severity of their global situation as it relates to phantom limb pain (as 
defined by each individual) compared to their baseline.  This scale has the words “very much worse” 
to the left by the number one, and “very much improved” to the right, adjacent to the number seven.  
The words “no change” are in the middle of the scale above the number four.  The Patient Global 
Impression of Change Scale has been validated in over ten prospective trials, including studies 
specifically involving peripheral neuropathy.  
 
 
 
 
Hypothesis 2b:  Physical and emotional functioning will be significantly improved 4 months 
following one cryoneurolysis procedure (as measured with the Brief Pain Inventory). 
 
 
 
It is well-recognized that, “pain is a complex, multidimensional, sensory, and emotional experience 
that is individually perceived and described in many different ways.”  This observation has led to 

 
 
Biomedical IRB Application Instructions 
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consensus recommendations that “multiple core domains and related measures be considered in 
pain treatment trials,” that “tap into a wider experience of pain over time and its impact on functioning 
and quality of life.”  Therefore, the proposed trial will include the Brief Pain Inventory, an instrument 
that includes—in addition to pain intensity scales—seven measures evaluating the pain’s interference 
with physical and emotional functioning, such as sleep, relations with others, and enjoyment of life.  
The Brief Pain Inventory has been used in countless clinical studies of chronic pain, and validated 
specifically in neuropathic pain states.  This instrument is associated with minimal subject burden and 
is easily interpreted by patients of all ages and education levels.  It has high test-retest reliability and 
correlates well with much longer questionnaires, including the McGill measures and EuroQol.  
 
 
 
 
Hypothesis 2c:  Depression will be significantly decreased 4 months following one 
cryoneurolysis procedure (as measured with the Beck Depression Inventory). 
 
 
 
In addition, multiple investigations demonstrate that factors such as anxiety and depression are 
strong predictors of pain intensity.  Therefore, the proposed study will evaluate additional 
psychosocial factors using the Beck Depression Inventory.  This 21-item instrument measures 
characteristic symptoms and signs of depression, requires only a 5th grade comprehension level to 
adequately understand the questions, and demonstrates high internal consistency (0.73-0.92, mean 
of 0.86), reliability and validity.  Each of the 21 factors is rated on a 0-3 scale, and then summed to 
produce the total score of 0-63.  Mild, moderate, and severe depression is defined with scores of 10-
18, 19-29, and 30-63, respectively.  While this instrument requires less than 10 minutes to complete, 
on average, it will be administered only at the initial baseline and four months following each 
treatment intervention (initial and crossover) as well as Month 12 to minimize subject burden and 
fatigue. 
 
 
Additional pain-related data.  Frequency and average duration of non-painful phantom sensations, 
phantom limb pain, and residual limb pain will be assessed.  In addition, supplemental analgesic use 
will be recorded, and other pain locations/severity will be evaluated using the NRS.  Lastly, to 
investigate masking adequacy, subjects will be queried on Day 0 following the treatment procedure 
on which treatment they believe they received (active cryoneurolysis vs. sham/placebo). 
 
Data collection.  Subject demographic and cryoneurolysis administration data will be uploaded from 
each enrolling center via the Internet to a secure, password-protected, encrypted central server 
(RedCap, Cleveland Clinic, Cleveland, Ohio).  The questionnaires for all subjects—regardless of 
enrolling center—will be administered by telephone from the University of California San Diego by 
research coordinators specifically trained in these instruments’ application, minimizing inter-rater 
discordance.  Staff masked to treatment group assignment will perform all assessments.  This web-
based data-collection protocol has been used successfully by the investigators for numerous 
previously published multicenter clinical trials.  Each data collection phone call will require 
approximately 15 minutes, with any time point including the Beck Depression Inventory requiring an 
additional 15 minutes. 
 
 
 
 
Statistical Plan and Data Analysis 

 
 
Biomedical IRB Application Instructions 
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The randomized groups will be descriptively compared on baseline demographic and pain variables 
using descriptive statistics.  In particular, groups will be considered well-balanced on a particular 
baseline variable if the standardized difference (difference in means or proportions divided by the 
pooled standard deviation) is less than
2 / n , where n is the per-group sample size.  The primary 
analysis will be modified intention-to-treat, in which all randomized subjects who received any of the 
study treatment will be included and retained in their respective treatment groups.  
 
Aim 1: Primary outcome.  We will assess the average causal effect of cryoneurolysis versus 
sham/placebo on phantom limb pain intensity (average pain over past 72 hours) at 4 months after the 
initial treatment using analysis of covariance to adjust for baseline pain intensity and any imbalanced 
baseline variables (see above).  Results will be summarized as the least squares difference in means 
at 4 months and 95% confidence interval. Mean and standard deviation change from baseline 
intensity will also be summarized. Similar analyses will be conducted for the secondary outcomes of 
current/present, worst and least phantom pain, as well as average and worst residual limb pain. 
 
Secondary outcomes.   
Aim 2a.  The randomized groups will be compared on the global measure of improvement (Patient 
Global Impression of Change Scale) at 4 months using the Mann-Whitney test. Proportional odds 
logistic regression will be used to adjust for any imbalanced baseline variables, as appropriate. 
Aim 2b.  The randomized groups will also be compared at 4 months on the seven measures of Brief 
Pain Inventory, which evaluate pain’s interference with physical and emotional functioning.  We will 
use a mixed effects multivariate model (random subject term, fixed treatment effect, unstructured 
correlation matrix) to first assess whether the treatment effect differs across the individual measures 
(i.e., treatment-measure interaction).  In presence of an interaction, each measure will be evaluated 
univariably.  Otherwise, an overall treatment effect will be estimated from the mixed effects model as 
the primary result for this aim. 
Aim 2c.  Analysis of covariance adjusting for baseline score will be used to assess the treatment 
effect of cryoneurolysis versus sham on depression at 4 months after randomization as measured by 
the Beck Depression Inventory.   
 
Blinding assessment.  To assess the quality of the subject blinding as to initial treatment 
assignment, we will ask each subject on Day 0 to speculate which treatment they received (actual or 
sham cryo). A Pearson’s chi-square test will be used to compare the proportion of correct 
speculations in each treatment group. 
 
For all analyses, alternative statistical methods will be used if the assumptions of the planned 
analyses are not met.  For instance, t-tests or regression analyses on the change or percent change 
from baseline (depending on which is less correlated with baseline score) will be used instead of 
analysis of covariance when comparing groups on the 4-week outcomes if the treatment group-by-
baseline interaction is significant.  Transformations of the data or Mann-Whitney test or other non-
parametric procedures will be used if the assumptions of normality and/or equal variances are not 
met.  
 
Crossover phase. Beginning 4-6 months after the original randomization, requesting subjects will 
receive the opposite treatment from that received in their original randomization, and the same 
measurements will be collected through 4 months (see Table 1).  This option will allow all subjects 
the opportunity to receive the study treatment.  Although a completely unbiased assessment of the 
average causal effect will not be possible for this phase because the second treatment will be 

 
 
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voluntary, and crossover will thus likely be requested more often from those receiving sham/placebo 
in the first phase, we will still descriptively report the average treatment effect from those choosing to 
cross over (but no testing will be done; unbiased average causal effect will be obtained from the first 
phase).  
 
More importantly, we will estimate the variability in the individual causal effects of cryoneurolysis 
versus sham/placebo using this crossover design. Variability of the individual causal effects cannot 
be directly estimated in a parallel group study (e.g., from the main portion (Aim 1) of this study we 
can only directly estimate the average causal effect), since only the outcome for the single treatment 
received is measurable for each subject.  However, estimation of the variability of the individual 
causal effects from the crossover study, quantified as the standard deviation of within-subject 
differences on treatment versus sham/placebo, will provide valuable information about the 
heterogeneity of the treatment effect across subjects associated with cryoneurolysis treatment of 
phantom limb pain. We will also use regression models to explore whether any baseline factors are 
associated with higher or lower causal effects of treatment. 
 
Long-term follow-up.   Data for all outcomes will also be collected at 12 months post randomization.  
Due to the crossover design, we will not be able to directly assess the treatment effect of 
cryoneurolysis versus sham on these outcomes.  Rather, we will descriptively assess the change 
from the initial baseline to 12 months for various groups of subjects: 1) all who received the active 
treatment either initially or in the crossover, 2) initial control subjects who were not crossed over; 3) 
initial control subjects who were crossed over; 4) initial treated subjects who were not crossed over; 
5) initial treated subjects who were crossed over. 
 
Dropouts.   At most, about 7% of subjects in each group are expected to drop out of the study before 
reaching the 4-month primary outcome assessment (based on an unpublished pilot study of pre-
emptive continuous peripheral nerve block use for surgical amputation, for which we observed 1 of 
15).  Since the current study will consist of volunteers traveling to the centers for enrollment and a 
treatment procedure, we expect even less.  For those missing 4-month data we will use the last-
observation-carried-forward method if the brief pain inventory was measured at 3 months. Otherwise, 
we will use intent-to-treat and conservatively assign the best observed score to the sham/control 
group and the worst score for the treated group subjects.  We do not expect any appreciable effect of 
dropouts on either the power of the study or the unbiasedness of study results.   
 
Interim analyses. We will conduct interim analyses to assess efficacy (rejecting null) and futility 
(rejecting alternative) at each 25% of the maximum enrollment using a group sequential procedure. 
Specifically, a gamma spending function will be used with parameters -4 and -2 for efficacy and 
futility, respectively.  Thus, boundaries at the 1st through 4th analyses for efficacy (futility in 
parentheses) will be P≤ 0.0016 (P>0.9572), P≤ 0.0048 (P>0.7186), P≤ 0.0147 (P>0.2389) and P≤ 
0.0440 (P>0.0440) (Figures below). 
 

 
 
Biomedical IRB Application Instructions 
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Type I error.  We will use a parallel gatekeeping procedure to control the study-wide type I error at 
0.05.  For this procedure, we therefore a priori prioritize the study outcomes into ordered sets, as Aim 
1, Aim 2a, Aim 2b and then Aim 2c. Analysis will proceed in that order, and testing will proceed 
through each “gate” to the next set if and only if at least one outcome in the current set reaches 
significance. The significance level for each set will be 0.05 times a cumulative penalty for non-
significant results in previous sets (i.e., a “rejection gain factor” equal to the cumulative product of the 
proportion of significant tests across the preceding sets). Within a set, a multiple comparison 
procedure (Bonferroni correction) will be used as appropriate to control the type I error at the 
appropriate level.  SAS statistical software (Carey, North Carolina), R programming language (The R 
Project for Statistical Computing) and East 5.3 software (Cytel Inc.) will be used for all analyses. 
 
Sample size considerations.  Our sample size estimate is based on the primary specific aim of 
whether the addition of cryoneurolysis decreases phantom limb pain intensity resulting from an 
amputation compared with current standard-of-care treatment at 4 months following cryoneurolysis.  
Receiver operating characteristic curve analyses demonstrate that changes from baseline of at least 
1.7 along a 10-point NRS accurately identified patients who rated improvements as “much improved” 
or more, compared with those who perceived no change or worsening following analgesic 
interventions.  Multiple additional studies confirm this degree of reduction as clinically meaningful to 
individual patients with chronic pain. Meaningful group differences in the mean change would be 
somewhat smaller than important changes for individuals. 
 
Therefore, we power our study to be able to detect group differences in mean change from baseline 
of 1.7 points or more on the NRS.  Based on a conservative standard deviation estimate for each 
group of 3.0 at 4 months, a correlation of 0.50 between baseline and follow-up NRS, a two-sided test 
at the 0.05 significance level, power of 0.90, and 4 equally spaced analyses (3 interim and 1 final, as 
needed), a maximum of 72 subjects in each group (N=144 total) is required (East 5.3 software, Cytel 
Inc).  The expected sample size for this group sequential design (i.e., average sample size over 
thousands of such trials, stopping when a boundary is crossed) is a total of 100 under the alternative 
and 102 under the null hypotheses. Table 2 reports boundary crossing probabilities at each of the 4 
analyses for this design, assuming that either the null or alternative hypotheses were true.  For 
example, there is a cumulative 8%, 37% and 75% chance of crossing a boundary at the 1st, 2nd and 
3rd analyses, respectively, if the alternative hypothesis were true. However, if the true standard 

 
 
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deviation at 4 months were smaller, say 2.5 instead of 3.0, then the cumulative probability of stopping 
for efficacy at the 1st, 2nd and 3rd analyses, respectively, would increase to 16%, 55% and 88% under 
the alternative hypothesis.    
 
Table 2.   P-value boundaries and boundary crossing probabilities for group sequential design 
Fraction  
of Maximum 
Accrual 
Cumulative 
Accrual 
Alpha 
Spent 
Beta 
Spent 
P-value 
Boundaries 
Boundary Crossing 
Probabilities 
H0 
H1 
Under H0 Under H1 
0.250 
36 
0.002 
0.010 
0.0016 
0.9572 
0.044 
0.083 
0.500 
72 
0.006 
0.027 
0.0048 
0.7186 
0.269 
0.290 
0.750 
108 
0.018 
0.054 
0.0147 
0.2389 
0.485 
0.379 
1.000 
144 
0.050 
0.100 
0.0440 
0.0440 
0.202 
0.248 
 
 
10. 
HUMAN SUBJECTS 
Approximately 60 subjects will be enrolled at UC San Diego, of approximately 200 participants at all 
sites. 
 
Inclusion criteria:  Adult patients of at least 18 years of age, (1) with a lower limb traumatic or 
surgical amputation at least 12 weeks prior to enrollment distal to the hip (femoral head remaining); 
(2) who experience at least moderate phantom limb pain—defined as a 3 or higher on the Numeric 
Rating Scale (NRS; 0-10, 0= no pain; 10=worst imaginable pain)—at least daily for the previous 2 
months. (3) accepting of a cryoneurolysis procedure; and, (4) willing to avoid both changes to their 
analgesic regimen as well as elective surgical procedures from 1 month prior to and at least 4 
months following the initial cryoneurolysis procedure. 
 
Exclusion criteria:  (1) allergy to amide local anesthetics; (2) pregnancy; (3) incarceration; (4) 
inability to communicate with the investigators; (5) morbid obesity (body mass index > 40 kg/m2); 
and, (6) possessing any contraindication specific to cryoneurolysis such as a localized infection at the 
treatment site, cryoglobulinemia, cold urticaria and Reynaud’s Syndrome. 
 
11. 
RECRUITMENT AND PROCEDURES PREPARATORY TO RESEARCH 
Study subjects will be identified by each of the enrolling centers from four sources: 
1.  Databases.  Databases will be examined by non-investigator database managers following 
Institutional Review Board approval, and subsequently queried quarterly.  The site directors will 
provide database managers with Institutional Review Board-approved letters describing the study 
with investigator contact information sealed in envelopes with postage applied.  Database managers 
will then use existing databases to identify individuals with a prior amputation and apply address 
labels to the supplied letters and mail them—this is to adhere to current HIPAA rules. Individuals who 
are interested in the study will be directed to contact the study investigators or research coordinators 
for a thorough description of the study purpose and protocol.  Of note, all U.S. service members with 
a limb amputation during Operation Iraqi Freedom and Operation Enduring Freedom are included in 
an amputee-specific database, regardless of inclusion in surgical or chronic pain databases; but, the 
information within this database is not available for clinical investigations to protect patient privacy.  
We will strive to reach all of these individuals using the additional methods described below to ensure 

 
 
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that all U.S. military personnel are informed of the study and have the opportunity to participate. 
2.  Clinics.  Each site director will identify and meet quarterly with clinic (amputee-specific, surgical, 
and chronic pain) personnel to both inservice them on the study protocol as well as provide 
Institutional Review Board-approved written information describing the investigation that may be 
given to prospective subjects during clinic visits.  Clinic patients will come into contact with their 
healthcare providers during regularly-scheduled clinic visits, and these providers will briefly describe 
the study and offer written information to patients with phantom limb pain who voice interest in the 
study.  Patients who are interested in the study will be required to give permission for a research 
coordinator to contact them to adhere to Health Insurance Portability and Accountability Act (HIPAA) 
requirements. 
3.  Advertisements.  The Primary Investigator will place Institutional Review Board-approved study 
advertisements within print and web-based publications that are frequently read by the target 
population.  Additionally, site directors will provide similar Institutional Review Board-approved 
advertising material to the leaders of local and regional amputee focus/support groups.  Individuals 
who are interested in the study will be directed to contact the study investigators or research 
coordinators for a thorough description of the study purpose and protocol.  In addition, Institutional 
Review Board-approved advertisements will be placed and a study-specific information page created 
within Facebook, which has over 1-billion members.  Of note, the United States Army, Navy, Air 
Force, and Marines all have dedicated Facebook information pages.  Information on how to volunteer 
for the study will also be placed in relevant Twitter feeds, such as the Clinical Trial Spotlight San 
Diego, and Clinical Connection, free portals for finding clinical trials.  Twitter users (over 300-million 
registered users) looking for trials will be able to access contact information to the study, and link to 
the Facebook page for additional information. 
4.  ClinicalTrials.gov.  The trial will be prospectively registered on the ClinicalTrials.gov website.  
One of the main purposes of trial registries is to provide the general population access to available 
ongoing investigations.  Contact information for the site directors will be included on the website so 
that prospective subjects may receive a thorough description of the study purpose and protocol.  
Which site director subjects contact is up to the subjects themselves, because all will be listed on the 
website.  However, for individuals desiring enrollment, site directors will suggest the optimal 
treatment center based on geographic location and military status (e.g., civilians can be treated 
exclusively at one of the civilian enrolling centers). 
Once a prospective subject contacts a site director or research coordinator by telephone or email, 
they will be provided information on the study purpose and protocol, as well as have any questions 
answered.  Inclusion/exclusion criteria will be reviewed and documented on a form to identify subject 
eligibility according to these criteria.  If a prospective subject is excluded for any reason, the reason 
for exclusion will be recorded, but no patient identifying information will be recorded.  Candidates who 
meet inclusion and exclusion criteria and desire study enrollment will be scheduled for their initial 
study treatment.  Written informed consent will be attained prior to any measurements or procedures 
the morning of the initial treatment (see below for a detailed description of the informed consent 
process). 
Recruitment of Active Duty Military Personnel.  The Chain of Command will not be involved in the 
recruitment of military personnel; and will in no way be asked to encourage or order soldiers to 
participate in the trial.  As per Department of Defense Directive 3216.2, an ombudsman will be 
present if any group briefings of Active duty personnel are scheduled, to help ensure that those 

 
 
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present understand that study participation is completely voluntary and will not influence their careers 
or standing within the military. 
Study Compensation.  To help compensate study subjects for their time and defray travel 
expenses, participants will receive $500 following each study treatment prior to discharge (initial and 
the optional crossover). We believe that this compensation amount is both fair and does not provide 
undue inducement.  The University of California San Diego Institutional Review Board approved 
similar amounts for subjects in clinical trials of similar time, risk and duration.  Currently, 24 USC 30 
limits payments to Active Duty military personnel for participation in research while on duty to blood 
donations (which are not required for the proposed investigation).  However, military personnel who 
are on official military leave status may receive compensation for study participation, and will do so at 
the same level and on the same schedule as described for civilians and Veterans.  
Recruitment and advertisement materials.  These materials are being developed and will be 
provided to the IRB prior to use.  Each of the 7 enrolling centers will include information specific to 
that center.  We believe that these documents accurately reflect the study and are not coercive or 
offer undue inducements. 
 
12. 
INFORMED CONSENT 
Once a prospective subject contacts a site director or research coordinator by telephone or email, 
they will be provided information on the study purpose and protocol, as well as have any questions 
answered.  Candidates who meet inclusion and exclusion criteria and desire study enrollment will be 
scheduled for their initial treatment.  Written informed consent will be attained prior to any 
measurements or procedures the morning of the initial treatment.  Each site director is responsible 
for ensuring that written, informed consent is obtained from every subject at his respective enrolling 
center.  Clinical research coordinators—also one for each enrolling site—will be specifically trained 
by the site directors to provide informed consent followed by documentation of informed consent 
using an Institutional Review Board-approved informed consent form.  When subjects present for 
their initial treatment, research coordinators will provide and attain written informed consent.  This will 
occur in private patient care rooms, so that subjects may feel comfortable asking questions of the 
research coordinator.  If a subject desires—or if there is a question that a research coordinator 
cannot answer—the site director will be called in by the research coordinator to discuss the study 
directly with the subject. 
We do not foresee any issues relevant to the mental capacity of the potential human subjects.  
Written, informed consent will be attained prior to any study procedures or measurements.  Following 
a history and physical by the site director, subjects will have an intravenous line inserted, external 
monitors placed, oxygen delivered by face mask, and conscious sedation provided with minimal 
intravenous fentanyl (opioid) and midazolam (benzodiazepine), when applicable (not all subjects 
require sedation).  This sedation is to increase the comfort of subjects during probe insertion.  
Therefore, subjects will not be sedated until following the written, informed consent process is 
completed. 
Subjects will be provided privacy and time for decision making both in the study 
description/explanation telephone call to the site director or research coordinator, as described 
above; and also the morning of the initial treatment using a private patient care room to again review 
the study, informed consent form, and answer any remaining subject questions.  As noted previously, 

 
 
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subjects may speak with the site director by telephone from initial contact through the morning of 
treatment; and, will have access during and following the treatment(s) with cellular phone and pager 
numbers provided upon discharge. 
This study protocol has follow-up data-collection telephone calls a maximum of 1 year following the 
initial study treatment, so repeated informed consent following the initial consent is unnecessary, as 
opposed to multi-year, longer-term clinical trials. 
Surrogate consent will not be accepted; therefore, if human subjects cannot provide consent on their 
own, they will not be offered study enrollment.  Consent by an individual’s Legally Authorized 
Representative is unacceptable for study enrollment.  Of note, minors (age < 18 years) will not be 
offered enrollment, as explained in Section 6b (Inclusion/Exclusion Criteria).  Therefore, assent will 
not be accepted during the informed consent process. 
 
13. 
ALTERNATIVES TO STUDY PARTICIPATION 
Potential study subjects may simply decline enrollment. 
 
14. 
POTENTIAL RISKS 
1. Infection.  There is the potential risk of infection since subjects will have a probe inserted through 
the skin.  Since there will be nothing left going through the skin or in the subject after the probe is 
withdrawn, the risk of infection is very small and there has never been a report of permanent 
injury due to infection following cryoneurolysis. 
2. Bleeding.  The probe does not have an open tip and is not particularly sharp, so there is a very 
low risk of having any type of bleeding as a result of treatment.  However, if it was to happen, we 
would hold pressure until the bleeding stopped. 
3. Falling.  The risk of falling due to cryoneurolysis in a lower extremity (leg) currently unknown, 
although it has never been reported in the medical journals.  Subjects will be provided with a pair 
of crutches following treatment if desired, although it is rare that an individual feels the need to 
use crutches to walk. 
4. The skin where the nerve is frozen could lose or gain color if the nerve is particularly close to the 
surface.  However, this has never been reported for deeper nerves and using the probe that will 
be used for this study. 
5. Since a nerve will be frozen, there is the chance of nerve injury.  However, in 5 decades of use, 
only a single case of “neuritis” (nerve irritation) has been reported in medical journals, and this 
went away after a few months. 
6. There is the risk of loss of confidentiality.  The following procedures will be done to maintain 
confidentiality:  written, paper forms will be kept in a locked medical office and the locked 
Investigational Pharmacy’s files.  Computerized records containing personal health information 
will be stored on password-protected and encrypted computers. 
15. RISK MANAGEMENT PROCEDURES AND ADEQUACY OF RESOURCES 
During the treatment, subjects will be continuously monitored with pulse oximetry, noninvasive blood 
pressure cuffs, and EKG (standard for catheter placement).  Subjects will receive an IV so that 
emergency medications could be given, if needed.  As described above, probes will be placed under 
sterile conditions as is standard-of-care for any percutaneous cryoneurolysis. 
 
Following treatment, the subjects will be contacted at least the day following treatment by an 

 
 
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investigator or research coordinator, and longer if the subject desires or if medically indicated (e.g., 
suspected possible infection).  Subjects will have a physicians’ pager and cellular phone numbers 
available to respond 24 hours/day and 7 days/week for at least the first week following treatment. 
 
The risks to confidentiality are the release of names/ telephone numbers/ demographic data (e.g. 
weight, age, height), which will be minimized by the use of password-protected computers and case 
report forms that will be stored in locked offices. 
 
Subjects will be given clear instructions to call an investigator with any questions or concerns 
regarding their study participation.  If a patient experiences an injury that is directly caused by this 
study, only professional medical care that they receive at the medical center.  No other compensation 
is offered.  Any adverse events will be reported to the IRB using the standard adverse events 
reporting and upon continuing review (depending on severity, as defined by the IRB). 
 
The study Data Safety Monitoring Board (DSMB) will be comprised of the Medical Monitor, a 
physician experienced in both clinical trial management and the ethical conduct of research, and a 
statistician, also well-experienced in multicenter trials.  All three of these individuals will be 
completely independent of the investigative team.  No member of the DSMB will have any financial, 
proprietary, professional, or other interests that may affect impartial, independent decision-making by 
the DSMB. The board will comprise individuals with no vested interest in the outcome of the research 
study.  The members will also sign a confidentiality statement.  The DSMB will operate from a charter 
describing its role, membership, reporting procedures, and meeting protocol. The DSMB will decide 
on its own protocols, set triggers for data review or analyses, and establish guidelines for monitoring 
the study, stopping the study for safety concerns, and for efficacy based on plans specified in the 
protocol.  Confidentiality will be maintained during all phases of DSMB review and deliberations.  
DSMB members will maintain strict confidentiality concerning all privileged trial results provided to 
them. The board will perform the following functions: 
 
• Approval of the trial protocol before enrollment of patients 
• Review the data in order to determine efficacy, futility, and safety, and to determine whether 
the study should continue 
• Review data quality and data integrity 
• Evaluate risk versus benefit by thorough examination of the data accumulated 
• Determine whether the trial is proceeding as planned, the protocol is being followed, the 
recruitment of patients is on schedule, and data are being collected with the proper 
accuracy 
• Review patient dropouts, if any, and make appropriate recommendations 
• Determine whether safety concerns have been raised by the experimental or control 
treatments 
 
Review Items.  Items to be reviewed by the DSMB include: 
• Interim/cumulative data for evidence of study-related adverse events 
• Interim/cumulative data for evidence of efficacy according to pre-established statistical 
guidelines in the study protocol 
• Data quality, completeness, and timeliness 
• Performance of individual centers 
• Adequacy of compliance with goals for recruitment and retention, including those related to 
participation of women and minorities 
• Adherence to the protocol 

 
 
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• Factors that might affect the study outcome or compromise the confidentiality of the trial data 
(such as protocol violations etc.) 
• Factors external to the study such as scientific or therapeutic developments that may impact 
participant safety or the ethics of the study 
 
Recommendations.  The DSMB will conclude each review with their recommendations to the 
principal investigator and primary site Institutional Review Board (at the University of California San 
Diego) as to whether the study should continue without change, be modified, or terminated. 
Recommendations regarding modification of the design and conduct of the study may include: 
• Modifications of the study protocol based upon the review of the safety data 
• Suspension or early termination of the study or of one or more study arms because of serious 
concerns about patients' safety, inadequate performance, or rate of enrollment 
• Suspension or early termination of the study or of one or more study arms because study 
objectives have been obtained according to pre-established statistical guidelines 
• Optional approaches for executive committee and investigators to consider when the DSMB 
determines that the incidence of the primary study outcomes is substantially less than 
expected, such as recommendations to increase the number of trial centers or extend the 
recruitment period 
• Corrective actions regarding a study center whose performance appears unsatisfactory or 
suspicious 
 
Appropriate reports will be made to the Institutional Review Boards at all enrolling centers and the 
executive committee, comprised of all co-investigators and site directors 
 
 
16. PRIVACY AND CONFIDENTIALITY CONSIDERATIONS INCLUDING DATA ACCESS AND MANAGEMENT 
Disposition of data.  The original, hard-copy signed informed consent forms and case report forms 
will be stored within the local site director’s locked office, where they will remain for at least 7 years.  
These hard copies will not be mailed or otherwise transferred. Data will be uploaded and stored in 
one location: the central servers of Department of Outcomes Research at the Cleveland Clinic, a 
department dedicated completely to clinical research. This department has a full-time Research 
Electronic Data Capture (REDCap) programmer dedicated to developing REDCap databases and 
providing support for clinical trials. REDCap is a relational database for data entry and auditing. This 
is a web-based application designed exclusively to support data capture for research studies.  The 
Department of Outcomes Research at the Cleveland Clinic web servers are encrypted and 
password-protected with multiple firewalls to the standards of the National Institutes of Health. Of 
note, the servers are backed up every night. In the case of a disk failure, only data written to the files 
since the last backup will be subject to loss and can be easily restored. Databases are protected 
through electronic measures using a multi-layered, but simple approach: all study related files will 
reside on the database server rather than on individual hard disk drives and the files will be protected 
by the operating systems features against general access. User names will be password protected.  
The electronic data will remain within the Department of Outcomes Research for 7 years following 
study completion.  The UCSD research coordinator may receive training at the UCSD CTRI in 
REDCap use. With such training, up to six hours of user support is provided without recharge. 
However, technical and most user support will be provided by the Cleveland Clinic. The USAMRMC 
(United States Army Medical Research and Materiel Command) is eligible to review study records at 
any time.  
Each local site will transfer certain PHI to the UCSD research coordinator who will make all data 

 
 
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collection phone calls for all subjects. PHI transferred will include the subject’s name, phone 
numbers, and study ID. This information will be transferred via a secure online system known as the 
Army Missile and Research, Development and Engineering Command Safe Access File Exchange 
System (AMRDEC SAFE). AMRDEC SAFE is a secure, password-protected, system that the military 
has approved, and requires, for the transfer of such data. Civilian centers may use this system if 
access is granted, or fax to a locked office with access restricted only to the UCSD study coordinator 
and the PI.   
Sharing study results. Following study completion, all subjects will be provided with the study 
results in written form and in language appropriate for non-medical individuals. In addition, the master 
dataset will be de-identified. 
 
17. POTENTIAL BENEFITS 
For subjects randomized to receive a sham treatment first:  There will be no difference between 
being in this study and deciding against participation.  Therefore, there is no potential for direct 
benefits from this sham “treatment”.  However, all subjects will be offered the option of participating in 
the cross-over arm of the study, in which case they would receive active treatment (see below). 
 
For subjects randomized to receive active cryoneurolysis first:  It is our hope that patients have 
a permanent decrease in their phantom limb and/or residual limb pain. 
 
Possible benefits to others:  Future patients may benefit if it is determined that cryoneurolysis 
decreases phantom limb and/or residual limb pain.  There are millions of individuals world-wide who 
suffer from these debilitating conditions, and finding an effective treatment would be a tremendous 
step forward in treating these individuals. 
 
18. RISK/BENEFIT RATIO 
Chronic phantom limb and residual limb pain cause significant disability for patients, and there is 
currently a dearth of reliable treatments for this debilitating pain.  Since infection and falling are the 
largest risks of this intervention, and there have no previous cases of permanent negative sequelae 
due infection or any falls reported in the literature, we believe the potential risks to be minimal 
compared to the potential benefits. 
 
Subjects will be given clear verbal and written instructions to call Dr. Ilfeld in the Department of 
Anesthesia at UCSD, with any questions or concerns regarding their study participation.  If a patient 
experiences an injury that is directly caused by this study, they will receive professional medical care at 
the University of California, San Diego. No other compensation is offered.  Any adverse events will be 
reported to the UCSD IRB using the standard adverse events reporting website and on continuing 
review (depending on severity, as defined by the IRB). 
 
19. EXPENSE TO PARTICIPANT 
There will be no additional costs to subjects as a result of being in this study, other than travel to and 
from the medical facility for the study treatment.  These expenses may include, but are not limited to, 
costs for fuel, bus service, parking, bridge tolls, and meals.  If a subject is injured as a direct result of 
participation in this research, the University of California will provide any medical care needed to treat 
those injuries. The University will not provide any other form of compensation for an injury. 
 

 
 
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20. COMPENSATION FOR PARTICIPATION 
To help compensate subjects for their time and to defray travel expenses, they will receive $500 
following each visit to the enrolling center, for up to $1,000 for subjects who undergo both an initial 
treatment and crossover treatment.  Currently, 24 USC 30 limits payments to Active Duty military 
personnel for participation in research while on duty to blood donations (which are not required for 
the proposed investigation).  However, military personnel who are on official military leave status may 
receive compensation for study participation, and will do so at the same level and on the same 
schedule as described for civilian and Veterans. 
 
21. PRIVILEGES/CERTIFICATIONS/LICENSES AND RESEARCH TEAM RESPONSIBILITIES 
Principal Investigator, Brian M. Ilfeld, MD, MS, is a board-certified anesthesiologist with fellowship 
training in and 17 post-training years experience with regional anesthesia and acute pain medicine.  
Dr. Ilfeld holds a license to practice medicine in California.  Dr. Ilfeld has medical privileges at the UC 
Medical Centers.  Dr. Ilfeld, or another investigator, will follow all subjects following their treatment.  
Dr. Ilfeld will be responsible for the overall management of this study, as well as for the well-being of 
study subjects. 
 
Co-investigators, Rodney Gabriel, MD, Matthew Swisher, MD, and John Finneran, MD, are board-
certified anesthesiologists with years of experience with regional anesthesia and acute pain 
medicine.  All hold a license to practice medicine in California and has medical privileges at the UC 
Medical Centers.  They will help consent subjects, perform a history and physical exam, perform the 
treatment on subjects, follow subjects following their treatment, and help manage the study (including 
regulatory work). 
 
The study will be overseen a medical monitor, Salim Hayek, MD, PhD, Case Western Reserve 
University; Cleveland, Ohio—in essence a study subject advocate.  As the Chief of Pain Medicine at 
Case Western Reserve University, Dr. Hayek is thoroughly experienced in management of clinical 
trials, the ethical conduct of clinical research, the patient population under investigation (amputees 
with phantom limb pain), and Pain Medicine interventions for chronic conditions (such as 
cryoneurolysis).  As such, Dr. Hayek is a strong study subject advocate.  Dr. Hayek has extensive 
experience working on committees and authoring/editing peer-reviewed evidence-based publications, 
and will be an active member of the DSMB.  He has been, and will continue to be, completely 
independent of the investigative team.  In addition, Dr. Hayek has no financial, proprietary, 
professional, or other interests that may affect impartial, independent decision-making by the DSMB. 
Lastly, he has no vested interest in the outcome of the research study. 
 
Baharin Abdullah is a research coordinator with the UCSD CTRI, with the required training—
including up-to-date CITI training—for her position. 
 
22. BIBLIOGRAPHY 
1.  Sherman et al: Prevalence and characteristics of chronic phantom limb pain among American veterans. Am J Phys Med 
1983; 62: 227 
2.  Hazelgrove et al: Phantom limb pain. Int J Low Extrem Wounds 2002; 1: 112 
3.  Nikolajsen et al: Phantom limb pain. Br J Anaesth 2001; 87: 107 
4.  Wartan et al: Phantom pain and sensation among British veteran amputees. Br J Anaesth 1997; 78: 652 
5.  Halbert et al: Evidence for the optimal management of chronic phantom pain. Clin J Pain 2002; 18: 84 
6.  Woolf et al: Neuropathic pain: Aetiology, symptoms, mechanisms, and management. Lancet 1999; 353:1959 
7.  Nikolajsen et al: Relationship between mechanical sensitivity & postamputation pain. Eur J Pain 2000;4:327 
8.  Flor et al: Phantom-limb pain as a perceptual correlate of cortical reorganization. Nature 1995; 375: 482 

 
 
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9.  Merzenich et al: Somatosensory cortical map changes following amputation. J Comp Neurol 1984; 224: 591 
10. Birbaumer et al: Effects of regional anesthesia on phantom limb pain are mirrored in changes in cortical reorganization. 
J Neurosci 1997; 17: 5503 
11. Ilfeld: Continuous peripheral nerve blocks. Anesth Analg 2011; 113: 904 
12. Schley et al: Continuous brachial plexus blockade in combination with the NMDA receptor antagonist memantine 
prevents phantom pain in acute traumatic upper limb amputees. Eur J Pain 2007; 11: 299 
13. Ilfeld, Preciado, Trescot: Cryoneurolysis for the treatment of sensory and motor peripheral nerves. Expert Rev Med 
Dev, In Press 
14. Trescot: Cryoanalgesia in interventional pain management. Pain Physician 2003; 6: 345 
15. Gage: History of cryosurgery. Semin Surg Oncol 1998; 14: 99 
16. Hsu et al: Reduction in muscular motility by selective focused cold therapy. J Neural Transm 2014; 121: 15 
17. Hsu et al: Wallerian degeneration and recovery of motor nerves.  Muscle Nerve 2015; 51: 268 
18. Moorjani et al: Effects of cryoanalgesia on post-thoracotomy pain and on the structure of intercostal nerves: a human 
prospective randomized trial and a histological study. Eur J Cardiothorac Surg 2001; 20: 502 
19. Moesker, Karl, Trescot: Treatment of phantom limb pain by cryoneurolysis of the amputated nerve. Pain Pract 2014; 14: 
52-6 
20. Dworkin et al: Research design considerations for chronic pain clinical trials. Pain 2010; 149: 177 
21. Cleeland et al: Pain assessment: Global use of the Brief Pain Inventory. Ann Med Singapore 1994;23:129 
22. Beck et al: An inventory for measuring depression. Arch Gen Psychiatry 1961; 4: 561 
23. Buckenmaier et al: Treating pain on the battlefield. Curr Pain Headache Rep 2010;14:1 
24. Buckenmaier et al: Continuous peripheral nerve block for battlefield anesthesia and evacuation. Reg Anesth Pain Med 
2005; 30: 202 
25. Plunkett et al: Supraclavicular continuous peripheral nerve block in a soldier. Br J Anaesth 2006; 97: 715 
26. Stojadinovic et al: Responding to challenges in modern combat casualty care. Pain Med 2006; 7: 330 
27. Baker et al: Battlefield anesthesia: advances in care and pain management. Anesthesiol Clin 2007;25:131 
28. Buckenmaier et al: Pain following battlefield injury and evacuation. Pain Med 2009; 10: 1487 
29. Bloomquist T: Amputation and phantom limb pain: a pain-prevention model. AANA.J 2001; 69: 211-217 
30. Ziegler-Graham et al: Estimating the prevalence of limb loss. Arch Phys Med Rehabil 2008; 89: 422 
31. Mayfield et al: Trends in limb amputation in the Veterans Administration. J Rehabil Res Dev 2000;37:23 
32. Stansbury et al: Amputations in U.S. military personnel in the current conflicts in Afghanistan and Iraq. J Orthop Trauma 
2008; 22: 43-46 
33. Lin et al: Evaluation of orthopaedic injuries in Operation Enduring Freedom. J Orthop Trauma 2004;18:300 
34. Nikolajsen et al: The influence of preamputation pain on postamputation phantom pain. Pain 1997; 72: 393 
35. Becker et al: Pain and health related quality of life in chronic non-malignant pain patients. Pain 1997;73:393 
36. Sherman et al: A survey of current phantom limb pain treatment in the United States. Pain 1980; 8: 85 
37. Sherman: Published treatments of phantom limb pain. Am J Phys Med 1980; 59: 232 
38. Sherman et al: Chronic phantom and stump pain among American veterans. Pain 1984; 18: 83 
39. Sherman RA: Phantom limb pain. Mechanism-based management. Clin.Podiatr.Med Surg. 1994; 11: 85-106 
40. Bleckner et al: Serum ropivacaine concentrations and systemic local anesthetic toxicity in trauma patients receiving 
long-term continuous peripheral nerve block catheters. Anesth Analg 2010; 110: 630 
41. Buckenmaier et al: Regional anesthesia in austere environments. Reg Anesth Pain Med 2003; 28: 321 
 
23. FUNDING SUPPORT FOR THIS STUDY 
Dr. Ilfeld is supported by a Department of Defense grant award (PR160263) which helps to support 
his nonclinical time and the product used in this investigation.  Start date for this grant will be 
September 2017 and termination date will be August 2021.  Please contact Brianne Decker, grants 
specialist, at (619) 543-4613 for information regarding this DOD grant. All funding for this 
investigation is provided by the Department of Defense. 

 
 
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24. BIOLOGICAL MATERIALS TRANSFER AGREEMENT 
Not applicable. 
 
 
25. INVESTIGATIONAL DRUG FACT SHEET AND IND/IDE HOLDER 
Not applicable since percutaneous cryoneurolysis and the products used for this protocol are all 
cleared by the United States Food and Drug Administration for use treating both acute and chronic 
pain.  Therefore, this is an on-label study. 
 
26. IMPACT ON STAFF 
Participants will be enrolled by investigators andresearch coordinators specifically hired and trained 
for the study. Subjects receiving treatment at UCSD will be seen at the CTRI. Since only the research 
center will be utilized, there will be no impact on hospital clinical staff. 
 
27. CONFLICT OF INTEREST 
The Department of Defense is funding this study in its entirety. There is no financial or otherwise 
conflict of interest for any of the investigators.   
 
28. SUPPLEMENTAL INSTRUCTIONS FOR CANCER-RELATED STUDIES 
Not applicable. 
 
29. OTHER APPROVALS/REGULATED MATERIALS 
None. 
 
30. PROCEDURES FOR SURROGATE CONSENT AND/OR DECISIONAL CAPACITY ASSESSMENT 
Not applicable:  surrogate consent will not be accepted.