Perception Threshold Tracking(PTT): A Novel Method for Early Detection and Grading of Diabetic Peripheral Neuropathy
Conditions: Neuropathy, Diabetic, Neuropathy, Painful
Sponsor: Aalborg University Hospital

Cover page for protocol 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sponsor-investigator name: 
Niels Ejskjær / Aalborg University Hospital 
Protocol number: 
VEK N-2019003 
Official title of study: 
Perception Threshold Tracking (PTT): A 
method for early detection and grading of 
diabetic peripheral neuropathy 
Short title: 
MEDON 
Document date: 
30.08.19 

Perception Threshold Tracking (PTT): A method for early detection and grading of diabetic peripheral neuropathy 
Protocol version: 2 
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Table of Contents 
I. PROJECT GROUP ............................................................................................................................................. 4 
II. BACKGROUND AND SIGNIFICANCE ................................................................................................................ 5 
A. Background ................................................................................................................................................ 5 
B. State-of-the-Art ......................................................................................................................................... 5 
III. STUDY OBJECTIVES ....................................................................................................................................... 6 
IV. METHODS ..................................................................................................................................................... 7 
A. Study Design .............................................................................................................................................. 7 
B. Study Population ....................................................................................................................................... 7 
C. Assessment of Resources .......................................................................................................................... 8 
D. Study Procedures ...................................................................................................................................... 8 
PainDETECT ................................................................................................................................................ 8 
Clinical examination................................................................................................................................... 9 
512 mN pinprick ........................................................................................................................................ 9 
Conventional nerve conduction ................................................................................................................ 9 
Corneal Confocal Microscopy .................................................................................................................... 9 
Quantitative Sensory Testing .................................................................................................................... 9 
Axon flair-mediated neurogenic vasodilation ......................................................................................... 11 
Perception Threshold Tracking ................................................................................................................ 11 
Multi-Level Magnetic Resonance Imaging .............................................................................................. 11 
Cuff occlusion .......................................................................................................................................... 12 
Blood samples .......................................................................................................................................... 12 
V. DATA COLLECTION ....................................................................................................................................... 13 
VI. DATA ANALYSIS .......................................................................................................................................... 13 
A. Sample Size Considerations ..................................................................................................................... 13 
B. Statistical Methodology ........................................................................................................................... 13 
VII. DATA AND SAFETY MONITORING PLAN .................................................................................................... 13 
VIII. STUDY LIMITATIONS ................................................................................................................................. 14 
IX. ETHICAL CONSIDERATIONS ......................................................................................................................... 14 
A. Informed Consent.................................................................................................................................... 14 
B. Risks and Side Effects .............................................................................................................................. 15 
C. Benefits to Subjects ................................................................................................................................. 15 
D. Costs to Subject ....................................................................................................................................... 15 
E. Compensation to Subject ........................................................................................................................ 15 

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F. Provisions for vulnerable subjects ........................................................................................................... 16 
X. PLANS FOR DISSEMINATION OF FINDINGS .................................................................................................. 16 
XI. REFERENCES ................................................................................................................................................ 16 
XII. APPENDICES   ............................................................................................................................................. 21 
PainDETECT .................................................................................................................................................. 21 
Consent form ........................................................................................................................................... 23 
Flowchart ................................................................................................................................................. 23 
 
 
 

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I. PROJECT GROUP 
 
Principle investigator 
Niels Ejskjær 
Professor of Endocrinology 
Steno Diabetes Center North Denmark (SDCN) 
Aalborg University & Aalborg University Hospital 
Email: n.ejskjaer@rn.dk 
Phone. +4597663656 
 
Work group 
 
Johan Bovbjerg Røikjer 
MD, Ph.D.-student 
SDCN & Department of Health Science and Tech-
nology (HST) 
Aalborg University & Aalborg University Hospital 
Suganthiya Santhiapillai,  
MSc, Ph.D.-student  
Department of Radiology and Endocrinology  
Aalborg University Hospital 
Jens Frøkjær 
Professor of Radiology 
Department of Radiology 
Aalborg University Hospital 
Carsten Dahl Mørch 
Associate Professor 
Center for Neuroplasticity and Pain (CNAP), SMI, 
HST 
Aalborg University 
Peter Vestergaard 
Professor of Endocrinology 
Head of Research SDCN 
Department of Endocrinology / SDCN 
Aalborg University Hospital 
Tine Maria Hansen, MSc, PhD, postdoc 
MRI Research Engineer 
Department of Radiology 
Aalborg University Hospital 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hjalte H. Andersen 
Assistant Professor 
SMI, HST 
Aalborg University 
Ryan Godsk Larsen 
Associate Professor  
Sport Science  
Aalborg University  
Lars Arendt-Nielsen 
Professor 
CNAP, SMI, HST 
Aalborg University 
Ole Kæseler Andersen 
Associate Professor 
CNAP, SMI, HST 
Aalborg University 
Merete Grothe Christensen 
Research Bio. Med. Lab. Technologist 
SDCN 
Aalborg University 
Morten Hasselstrøm Jensen 
MSc, Ph.D. 
SDCN 
Aalborg University Hospital 
 
 

II. BACKGROUND AND SIGNIFICANCE 
A. Background 
Diabetes Mellitus (DM) is a growing healthcare problem worldwide[1] and currently affects more than 451 
million people[2]. DM is associated with excess morbidity and mortality[3][4] with diabetic peripheral neu-
ropathy (DPN) being the most common of the classic diabetic complications[5]. This means, that half the 
diabetic population will develop DPN during their lifetime[6]. Moreover, DPN is also the leading cause for 
disability in diabetic patients, mainly due to its complications including autonomic disturbances and events 
related to the diabetic foot. The most important being diabetic foot ulcers, foot amputations, and the Charcot 
neuroarthropathy (CN), while approximately 20-30% of patients will suffer from invalidating neuropathic pain 
at some point during their lifetime[7]. Furthermore, diabetic foot ulcers have been associated with ischemic 
heart disease (IHD), which is the leading cause of premature mortality in patients with DM[8]. At the same 
time, the five-year mortality after new-onset diabetic ulceration have been reported to be 43-55%, and 74 % 
in patients with diabetic foot amputation, which is significantly higher than the five-year mortalities for many 
forms of cancer[9]. 
  Despite this, our knowledge is quite limited when it comes to risk stratification of the diabetic patients. It is 
clear that DPN is the single most important risk factor for development of complications regarding the dia-
betic foot whether talking about foot ulcers[10], amputations[11], or CN[12]. However, the current clinical 
tests to detect DPN are unspecific and with questionable sensitivity reported as low as 0.53[13]. Furthermore, 
the current clinical tests are solely assessing large fiber neuropathy and are not able to do a meaningful 
grading of the severity of the neuropathy, hence the need for new methods to provide efficient clinical out-
come measures and useful clinical endpoints. 
 
B. State-of-the-Art 
Diabetic peripheral neuropathy (DPN) is the most common complication to advanced diabetes mellitus 
(DM)[14], and is strongly associated with invalidating and life-threatening complications like foot ul-
cers[15][16], CN [17][18], painful neuropathy[19], risk of fall and fracture[20], and lower foot amputa-
tions[11]. The incidence of DPN is reported with great variance, but several large studies have reported num-
bers around 20% for DM type I[21][22][23] and 50 % for DM type II[24]. Furthermore, patients with foot 
pathology attributable to DPN exhibit significantly higher mortality rates than those solely attributable to 
vascular diseases. This applies to both non-infectious neuropathic foot ulcers[25] and acute CN [26].  
  At this point no treatment for the underlying nerve damage of DPN is available[27], and the pathogenesis is 
still not fully understood[28][29][30]. Therefore, current strategies focus on prevention of the initial nerve 
damage using annual screening-tests targeting large nerve fibers. Available tests for large-fiber function in-
clude vibration perception, proprioception, 10-g monofilament, and ankle reflexes[31]. Tests for small-fiber 
function are not routinely used, but include pinprick and temperature sensation[31]. Most studies have re-
ported that early stages of DPN mainly affect the small nerve fibers[32][33] raising the question of why these 
are not used in standard screening. However, mixed results do exist, as some studies have reported simulta-
neous development of DPN in small- and large nerve fibers[34][35]. The most common clinical tests (mono-
filament) have recently been reviewed, showing low sensitivity when trying to detect early neuropa-
thy[13][36], but wielding fine results when detecting severe neuropathy[37].  

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  In recent years several new tests for DPN has emerged. Most notably Neuropad[38][39], NeuroQuick[40],  
and Vibratip[41], alongside the NC-stat DPNCheck[42] for automated nerve conduction study(NCS), and SU-
DOSCAN[43] for a quantitative measurement of sweat function. Furthermore, more equipment-heavy diag-
nostic tools have been used in an increasing degree in settings of clinical research. These mainly include skin 
biopsies[44][45], corneal confocal microscopy (CCM)[46][45], and Quantitative Sensory Testing 
(QST)[47][48], which is combined test examining the sensory perception after application of different me-
chanical and thermal stimuli of controlled intensity evaluating the function of both large and small nerve 
fibers. Neuropad is an indicator test for measuring sweat production as an expression of small nerve-fiber 
function. The test is originally designed to detect neuropathy by changing color in accordance to sweat pro-
duction. However, some groups have also measured absolute time to color change suggesting this as a meas-
urement for severity of neuropathy[49]. The sensitivity and specificity of the test has been reported with 
great variance ranging from 65.1-100% and 32-78.5% respectively[50]. NeuroQuick is a small portable fan 
with ten different velocity settings testing thermal perception. It has been suggested as an early measure for 
neuropathy, but clinical trials are currently lacking. Vibratip and Ipswich Touch Test (IpTT)[51] both test pe-
ripheral vibration threshold and has proven consistent with established standards[41]. The NC-stat 
DPNCheck is an automated Nerve Conduction Study (NCS) aiming to increase the utilization of traditional 
NCS in a primary care setting. It has been reported to yield a sensitivity of 92% and a specificity of 82 % 
referenced against traditional NCS[52]. Tactile Circumferential Discriminator (TCD)[53] (cylindrical disc with 
metallic rods) and steel ball-bearing[54] both test for loss of protective sensation (LOPS). However, clinical 
use is limited due to lack of either sensitivity or specificity respectively. Like Neuropad, SUDOSCAN is designed 
to evaluate sweat secretion as a marker for small nerve-function and like Neuropad, SUDOCAN has been 
proposed as a tool for grading neuropathy[55]. In present studies, SUDOSCAN seems to correlate with vibra-
tion perception threshold as measured with a biothesiometer, while detecting DPN with a sensitivity of 78% 
and a specificity of 92%[56]. Axon reflex-mediated neurogenic vasodilation is another technique proposed as 
a method for early detection of DPN[59]. The method uses lacking vasodilatory response to cutaneous heat-
ing as a measurement of pre-clinical small fiber dysfunction, but currently remain on experimental 
ground[60][61]. In recent years, Magnetic Resonance Imaging (MRI)-scans with experimental sequences in-
cluding blood oxygen level dependent signal (BOLD)-[57] and track-density imaging (TDI)-MRI[58] have also 
been of interest, as these might be able to detect early neuropathy. However, further studies are needed. 
Lastly, an experimental technique for tracking membrane potential and excitability called Perception Thresh-
old Tracking (PTT)[62] has been suggested as a method for early detection of DPN, but the method needs 
clinical studies. 
 
III. STUDY OBJECTIVES 
To test Perception Threshold Tracking (PTT) and multi-level MRI as methods for early detection and grading 
of severity of neuropathy in patients with diabetes mellitus. 
To use multi-level MRI for characterization of peripheral and central nervous system changes related to neu-
ropathy in patients with diabetes mellitus. 
To compare Perception Threshold Tracking (PTT) and MRI to methods of traditional clinical practice and es-
tablished methods of clinical research like Corneal Confocal Microscopy (CCM) and Quantitative Sensory 
Testing (QST) as well as axon flair-mediated neurogenic vasodilation (AF). 
To test reliability of different measurements of neuropathy. 

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A better understanding of the underlying neuropathy of diabetes and new methods for early, reliable detec-
tion of DPN could result in intensified treatment and prophylactic measures prolonging of the complication-
free period of diabetes mellitus. Ultimately, this could potentially result in fewer complications related to the 
diabetic foot, and maybe even prolonged overall-survival and reduced healthcare cost. 
 
IV. METHODS 
A. Study Design 
This study is a prospective, observational, cross-sector study starting March 1, 2019 running until December 
1, 2021. The study will be conducted on patients from Aalborg University Hospital. Participants will be in-
formed about the trial both written and vocally and no changed in antidiabetic medication will be performed. 
The instruments will include Corneal Confocal Microscopy (CCM), Quantitative Sensory Testing (QST), axon 
flair-mediated neurogenic vasodilation (AF), handheld heart rate measurement device (VagusTM), cuff, and 
MRI, which are all established methods of clinical research. Furthermore, participants will be tested using 
monofilament and Biothesiometry, which equals actual clinical practice, and with Perception Threshold 
Tracking (PTT), which is a new take on an older, well-known technology. Further tests will include a handheld 
pinprick device, measurements of peripheral arterial disease (PAD) and conventional NCS. The main endpoint 
of the study will be sensitivity/specificity of all examinations, as well as a comparison of the newer methods 
against more well-established modalities.  
 
B. Study Population 
The study population include four well-defined groups of subjects: 
1. Patients with Type 1 diabetes and painful diabetic peripheral neuropathy (DPN).  
2. Patients with Type 1 diabetes and non-painful diabetic peripheral neuropathy (DPN)  
3. Patients with Type 1 diabetes and without diabetic peripheral neuropathy (DPN).  
4. Healthy control subjects matched for age, BMI, and gender. 
Diabetes duration, HbA1c, insulin use, ethnicity, and co-morbidities will be matched between group 1, 2 and 
3 to best ability. 
Inclusion criteria: 
1. Men and women minimum 18 years of age and maximum 70 years of age 
2. Signed informed consent form 
3. Diagnosed with diabetes type I (for group 1-3) 
4. Diagnosed with DPN defined as a threshold above 25-volt biothesiometry or absent feeling on the 
big toe using 10g-monofilament. (for group 1-2) 
5. Answered questionnaire: PainDETECT 
6. Nothing abnormal on initial tests (group 4) 
7. Accepted initial screening blood samples 
8. MRI-compatible participant 
Exclusion criteria: 
1. Current or previous alcohol- or drug abuse  

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2. Abnormal screening blood samples 
3. Not being able to understand Danish written and/or verbally 
4. Not being able to corporate to examination (e.g. not being able to speak, suffering from senile de-
mentia etc.)  
5. Previous chemotherapy or intake of experimental medicine 
6. Active HSV- or VZV-infection or known HIV 
7. Known severe skin disease 
8. Known neural damage or disease in the neural system (e.g. MS, Guillain-Barre etc.) 
9. Critical limb ischemia defined as in current clinical consensus[63] 
10. Allergy or intolerance to histamine or inability to make do without for one day 
11. Pregnancy 
12. Active cancer-disease 
 
C. Assessment of Resources 
The study will be conducted in collaboration with Steno Diabetes Center Northern Denmark (SDCN) and De-
partment of Radiology, Aalborg University Hospital. SDCN has access to patients with diabetes and can pro-
vide the necessary premises.  
The study will be conducted in collaboration with Center for Neuroplasticity and Pain (CNAP), Aalborg Uni-
versity and SDCN, who have the technical and financial resources needed to establish the setup and conduct 
the examinations. Furthermore, an experienced research Bio. Med. Lab. Technologist is signed for the project 
thereby, alongside the Ph.D.-students, ensuring trained personnel. The study is set for a long inclusion, but 
additional time is available if required, as the Ph.D.-students are employed until 31st of October 2022. 
The project is initiated by Professor Niels Ejskjær, Aalborg University and SDCN, and is supported economi-
cally by Steno Diabetes Center North Denmark and Aalborg University. The support is however limited to 
staff salary and purchase of smaller equipment. 
D. Study Procedures 
Diabetic patients will be recruited during their scheduled visit to the outpatient clinic of SDCN, Aalborg Uni-
versity Hospital. Healthy controls will be recruited based on the included diabetic population using relatives 
of diabetic patients, medical students, and people known by participating staff.  
After the informed consent is obtained the screening of the participant will be executed. Afterwards the 
participant have to attend three different sessions as shown in appended flowchart. Each session includes 
different tests and the sessions will be ordered randomly, since they have no influence on each other. More-
over, a conventional nerve conduction test will be performed, however this test will be performed as per 
usual by the department of neurophysiology, as this is a standard examination for patients with neuropathy.  
 
PainDETECT 
PainDETECT is an English questionnaire validated for detecting neuropathic pain[64]. It has later been trans-
lated and validated in Danish[65], and will be given each participant alongside instructions after form of con-
sent has been signed. The patients will answer the questionnaire individually and will need to complete this 
before any other examinations can be conducted, as this will help placing the patients in the correct group. 
Estimated time: 5-15 min.   

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Clinical examination  
This mainly include biothesiometry and 10g-monofilamnent as these simple measures equals current clinical 
standard when screening for neuropathy. In biothesiometry, the detection limit of vibration is determined 
with slow rising strength until the patient perceives the vibration. In 10g-monofilament testing the physician 
uses a thin nylon fiber, which bends at a pressure of 10 grams. The filament is applied to four specific spots 
at the bottom of the foot. The tests are usually conducted during the scheduled visit to the outpatient clinic. 
If not, these tests will be performed just after inclusion before other experimental tests. This point also in-
cludes measurements of blood pressure measured at the arm, leg and toe as well as oxygen pressure in the 
blood vessels of the toe (TPO2). This allows a good assessment of potential PAD. Results from the clinical 
examination might be collected as a Neuropath Disability Score (NDS). Above mentioned examinations might 
be conducted at any of the later mentioned sessions (see appended flowchart). 
Estimated time: 15-20 min (possibly partly conducted during already scheduled appointment). 
 
512 mN pinprick 
This is a small custom-made pinprick device based on standardized QST (see below) principles. The device 
functions much like the monofilament described above but differs since it has a sharp finish thereby activat-
ing not large- but small nerve fibers. This test is conducted either during the scheduled visit or just after 
inclusion in the study alongside clinical examination. 
Estimated time: 5 min. 
 
Conventional nerve conduction 
Will be conducted by the department of Neurophysiology, Aalborg University Hospital, as “golden standard” 
for assessment of large nerve fiber function. The test is a standard clinical test and might already be con-
ducted at the time of inclusion.  
Estimated time: 30 min. 
 
Corneal Confocal Microscopy 
Corneal Confocal Microscopy (CCM) is a noninvasive method for studying of the human cornea in vivo. It is 
used in an increasing fashion for assessment of corneal small nerve fiber pathology as an expression of pe-
ripheral neuropathy[46]. Before examination, the eye of the participant will be anaesthetized using 0.4% 
benoxinate hydrochloride and Viscotears for front eye lubrication. The examination will be conducted by 
personnel trained by seasoned international operators.  
Estimated time: 60 min. 
 
Quantitative Sensory Testing 
Quantitative Sensory Testing (QST) is a way of assessing large and small sensory nerve fiber function using 
several different stimuli. The method has been verified using different protocols, and will be conducted in 

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accordance to the original protocol by German Research Network on Neuropathic Pain (DFNS)[66]. All meas-
urements are made on the dorsum of the foot. The test will be conducted by either PhD-students or research 
bio. med. lab. Technologist. The protocol consists of seven tests measuring 13 parameters including: 
 
1) Thermal detection, thermal pain thresholds and paradoxical heat sensations:  
The tests for thermal sensation will be performed using a thermal sensory testing device. Cold de-
tection threshold (CDT) and warm detection threshold (WDT) are measured initially and the number 
of paradoxical heat sensations (PHS) are counted. Cold pain threshold (CPT) and heat pain threshold 
(HPT) are then calculated as a mean threshold temperature of three consecutive measurements. All 
thresholds are to be obtained with ramped stimuli increasing by 1 degree Celsius per second, termi-
nated as the patient presses a button. Cut-off temperatures are set at zero and 50 degree Celsius 
and the baseline temperature is set at 32 degree Celsius, as this represents the mean value of skin 
temperature.  
 
2) Mechanical detection threshold for modified von Frey filaments 
Mechanical detection threshold (MDT) is to be measured using a standardized set of modified von 
Frey hairs that exert forces between 0.25 and 512 mN. The final threshold will be the geometric mean 
of five series of ascending and descending stimulus intensities. 
 
3) Mechanical pain threshold for pinprick stimuli 
Mechanical pain threshold (MPT) is to be measured using thin, blunt, weighted pinprick stimulators 
that exert forces between 8 and 512 mN. The set will consist of seven different stimulators and the 
MPT will be the mean of five series. 
 
4) Mechanical pain sensitivity and dynamic mechanical allodynia 
Mechanical pain sensitivity (MPS) is to be tested using the same weighted pinprick stimuli as for MPT 
(see above). To obtain a stimulus–response-function, the seven pinprick stimuli is applied in a bal-
anced order, five times each, and the subject is asked to give a pain rating for each stimulus on a 0–
100 numerical rating scale with 0 being no pain and 100 being worst pain imaginal. 
Stimulus–response-functions for dynamic mechanical allodynia (ALL) are determined using a set of 
three light tactile stimulators: a cotton wisp (force: 3 mN), a cotton wool tip fixed to an elastic (force: 
100 mN), and a standardized brush (force: 200–400 mN). The three tactile stimuli are applied five 
times each with a single stroke of approximately 1–2 cm in length over the skin. They are intermin-
gled with the pinprick stimuli in randomized order and subjects are asked to give a rating on a scale 
equal to the one used for MPS (see above). 
 
5) Wind-up ratio: the perceptual correlate of temporal pain summation for repetitive pinprick stim-
uli 
In this test of temporal summation, the perceived magnitude of a single pinprick stimulus is com-
pared with that of a train of 10 pinprick stimuli of a force of 256 mN repeated at a rate of one prick 
per second. Subjects are asked to give a pain rating representing the pain at the end of the train using 
the same scale as for MPS and ALL (see above). Single pinprick stimuli are alternated with a train of 
10 stimuli until both are done five times at five different skin sites on the same foot. The mean pain 
rating of trains divided by the mean pain rating to single stimuli is calculated as wind-up ratio (WUR) 
 

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6) Vibration detection threshold 
Vibration detection threshold (VDT) is tested using a tuning fork (64 Hz) placed over the medial mal-
leolus. Vibration threshold is determined by the mean of three series of descending stimulus inten-
sities. 
 
7) Pressure pain threshold 
This test is performed using a pressure gauge device able to deliver a pressure up to 200 N/cm2. The 
pressure pain threshold (PPT) is determined with three series of ascending stimulus intensities on m. 
tibialis ant., each applied as a slowly increasing ramp of 50 kPa/s. 
Estimated time: 60-90 min. 
 
Axon flair-mediated neurogenic vasodilation 
Axon flair-mediated neurogenic vasodilation (AF) uses Laser Doppler flowmetry (LDF) [66] or laser Doppler 
imaging (LDI) [67] to analyze vasomotor small fiber function by quantifying the integrity of the vasomotor-
mediated axon reflex. While LDF assesses the flare response following acetylcholine iontophoresis with tem-
poral resolution at a single defined skin point, LDI records flare responses with spatial and temporal resolu-
tion, generating a two-dimensional map of superficial blood flow [61]. In this study, the participants’ surface 
skin temperature of the dorsum of the foot will be standardized to 32 degree Celsius using a warm blanket 
or warming pad. Either histamine or Acetylcholine will then be applied to a small area and vasodilation will 
be measured in accordance to earlier studies[67].  
Estimated time: 15-20 min 
 
Perception Threshold Tracking 
Perception Threshold Tracking (PTT) is a potential new method for early detection of neuropathy. It is a fur-
ther development of conventional threshold tracking[68] that excels in describing membrane potential of 
not only large, but also small nerve fibers[62]. The trial is conducted using two types of surface electrodes 
placed on the dorsum of the foot: patch and pin electrodes for stimulation of large and small nerve fibers 
respectively. The perception threshold is then estimated by slowly increasing the intensity of stimuli until the 
patient presses a button indicating that the stimuli is perceived. As this happens, the intensity is initially 
heighted by 20 % and then lowered until the patient relieves the button indicating that the stimulus is no 
longer felt. The intensity is then lowered by 20% and then increased until perception is indicated by pressing 
the button. This is repeated three to five times to increase precision. The perception threshold is taken as 
the average of the six to ten times the patient pressed the button. This procedure will be repeated a number 
of times using different electrodes. The patient will feel no significant pain, as the stimuli given is just around 
the individual perception threshold, which is a huge benefit of this method compared to other available neu-
ral tests.  
Estimated time: 35-45 min 
 
Multi-Level Magnetic Resonance Imaging 
The multi-level MRI will be used to assess brain structure, function and metabolism and the peripheral nerve 
structure and function. The MRI session will consist of 1) an anatomical 3D scanning of the brain, 2) functional 

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MRI scan, 3) whole brain diffusion tensor imaging (DTI) for assessment of microstructure and fiber tracts, 4) 
MR spectroscopy assessment of metabolite, 5) BOLD signal for microvascular function in central level and 
peripheral level, and 6) MRI neurography of the peripheral nerve fibers. Additionally, cuff occlusion proce-
dure will be performed in the MRI scanner (cf. next section). Heart rate, respiration rate, will be recorded 
throughout the MRI scan. 
Estimated time: 90 min 
 
Cuff occlusion 
During the BOLD-MRI a cuff occlusion procedure will be performed to investigate the microvascular func-
tion. A cuff will be mounted around the leg, proximal to the knee, and inflated (240 mmHg) for 5 minutes to 
reduce or blockage the blood flow in the lower leg.  
Estimated time: 15 min  
 
Cognitive Tests  
A validated cognitive questionnaire will be performed to test the mild cognitive impairment and possible 
affected cognitive domains in the participants. Moreover, a cognitive task will be performed in the scanner 
in order to test the participants’ cognitive psychomotor speed and memory. 
Estimated time: 15-20 min 
 
Vagus Test Procedure 
A commercial available handheld device (VagusTM, Medicus Engineering Aps, Aarhus N, Denmark) will be 
used to test the autonomic nervous system and hereby the autonomic neuropathy. The participant will 
hold the device during four steps, where the device will 1) measure the heart rate at rest, 2) measure the 
heart rate response from laying position to standing position, 3) measure the relationship between heart 
rate during expiration and inspiration, and 4) measure the heart rate conditions during exhalation with a 
resistance of 40mmHg and at rest. 
Estimated time: 10-15 min 
 
Blood samples 
Blood samples will be taken after informed consent has been signed. Blood taken is estimated not to exceed 
15 ml and will include: 
Hgb, leukocytes and diff. counting, blood platelets, cobalamin, folate, sodium, potassium, creatinine, bicar-
bonate, CRP, ALAT, alkaline phosphatase, LDH, bilirubin, albumin, coagulation factors, vitamin D 1.25, Cal-
cium-ion, TSH, HbA1c and M-component.  
The blood samples will be taken and handled as usual by the biochemical department and will be destroyed 
after usage. No biobank will be made. All samples will start being analyzed immediately (within 2h), however, 
some samples (like vitamin D) will take some time to analyze. All results will be available within 10-12 days 
but samples will always be destroyed as soon as the analysis has been initialized. 

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V. DATA COLLECTION 
Patient data will be collected using the electronic patient journal (EPJ) after consent of the participant. Only 
patients with an active continuity of care at SDCN will be included (as well as healthy controls). The principle 
investigator is employed at SDCN as a medical doctor alongside one of the Ph.D.-students. 
Data collected will include demographic data such as name, age, sex, weight, and height, and clinical infor-
mation related to diabetes, such as type, duration, medication, complicating peripheral arterial disease, and 
status of complications. This to ensure correct grouping of the patients and to make sure that they meet the 
inclusion criteria. 
 
VI. DATA ANALYSIS 
A. Sample Size Considerations 
The following sample size estimation is made on the ability to separate a group of diabetic patients without 
neuropathy from a group of diabetes patients with ‘moderate’ neuropathy. The calculations are based on 
published measurement of the sensory nerve action potential amplitude in nerve fiber conduction assess-
ments[45]. Setting the significance level to 0.05 and the power to 0.8 results in a sample size estimation of 
16 persons in each group. 𝑛=
15.7∗𝜎2
𝑑2
=
15.7∗(4.72+7.52)
(14.4−5.7)2
= 16. The study involves several experiment 
measures, but we expect that max 20% of the measures will be dropouts. The number of participants needed 
to be recruited in each group will therefore be 𝑁=
𝑛
1−𝑑𝑟𝑜𝑝𝑜𝑢𝑡= 
16
0.8 = 20. We want to compare four groups 
as defined in section IV.B. Study Population. The total number of participants will therefore be 80.   
 
B. Statistical Methodology 
The data will initially be analyzed by a series ANOVA test to compare each measurement between the four 
groups.  
To avoid ‘mass significance’ a combined analysis of all measurements will be performed by logistic regression 
models (LRM) in three steps. This will allow an estimate of the odds ratio and accuracy of each of the meas-
urements and the measurements in combination. First, a series of univariate LRM will be performed for each 
measurement to investigate the odds ratio and accuracy of the individual. Second, a multiple factor LRM with 
all measurements as factors will be performed to estimate the odds ratio and accuracy for all measures in 
combination. Thirdly, factor reduction will be performed to assess the accuracy and odds ratio of a set of 
independent measures.  
Simple bivariate correlation will be performed to further investigate the relation between measurements. 
Due to the complexity of the measurements a further analysis of the interdependency of the measures using 
factor analysis will be performed.  
 
VII. DATA AND SAFETY MONITORING PLAN 
The project will be reported to the Data protection Agency (DPA), and all sensitive data will be stored in either 
Red-CAP or on the protected server of Aalborg University Hospital, “Elektronisk system adgang” (ESA). 

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Data are stored in accordance with the stipulations in The Danish Personal Data Protection Act (Persondata-
loven) and other relevant Danish legislation. If any data processing would occur in foreign countries, the data 
will be anonymized and handled according to the Danish Data Protection Act. 
The data can only be used for the interpretation of this project and will therefore not be of interest to third 
party.  
The project is included in “Region Nordjyllands fællesfortegnelse”. 
VIII. STUDY LIMITATIONS 
Both PTT and QST require corporation from the patients and include subjective measures of pain. As the 
measured thresholds are not purely objective, psychosocial factors might influence test results. 
The study includes diabetic patients exclusively and might not be applicable on populations with different 
causes of neuropathy. 
The expected number of participants are set at 80, thus maybe require additional larger studies to confirm 
findings.  
 
IX. ETHICAL CONSIDERATIONS 
A. Informed Consent 
The initial enquiry about participating will be made by the patients’ doctor during the scheduled visit at the 
outpatient clinic. The participants will then receive information regarding the project both written and ver-
bally and information for participants (“deltagerinformation”) will be handed out. Further information will 
later be given by a person fully informed about the study and with comprehensive knowledge of the different 
tests involved. Desirably either by the affiliated research bio. Med. lab. Technologist, the principle investiga-
tor, or one of the Ph.D.-students. 
Healthy control subjects will be approached verbally or in writing and will later receive information equal to 
the diabetic population. In other words, the healthy control subjects will first receive brief information along-
side information for participants (“deltagerinformation”). At least 24h after receiving written information, 
the healthy control subjects will be invited to participate in an informational meeting. If they chose to partic-
ipate, they will need to sign informed consent and proceed in the study like the other groups. 
The given information will include: 
 
That this is a scientific project testing a potential future method to detect diabetic peripheral neu-
ropathy. 
 
Information regarding the different tests involved incl. the risk of minimal to moderate pain associ-
ated with especially QST and cuff occlusion. 
 
That withdrawal from the project is possible at any time. 
 
That data collected will be published in an anonymized form, without any traces of personal data 
 
That data collected will be safely stored in accordance with the rules in force. 
Potential participants are allowed at least 24 hours for reflection and additional, informational meetings 
might be scheduled. During the scheduling of the informational meeting the participant will be informed 
about the right to an assessor or relatives both for the meetings and sessions. Participants are informed 
about their right and the possibility for an additional meeting before signing informed consent. No external 

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funding has been given. Neither of the core members have any conflicts of interest to declare. The project 
will be prospectively registered to www.clinicaltrials.gov.  
B. Risks and Side Effects 
As the project is mainly observational and contains no interventions the overall risk for the patient is ex-
tremely limited. No changed/adjustments in medication will be made and there will be no risk for hypo/hy-
perglycemia. The main issue for the participants is the fact that some time is needed for the conduction of 
the examinations, and the fact that patients might be required to take additional trips to the hospital. Fur-
thermore, a small amount of transient pain might be felt during some of the examinations; however, no 
lasting side effects are reported regarding any of the examinations. Listed below are the potential risks and 
side effects of the different examinations included in the protocol: 
1. Blood samples: Almost negligible risk of infection, small bleeding or pain around syringe in-
sertion. 
2. QST/PTT: Participants might feel a small amount of transient pain during some parts of the 
examinations. No lasting pain will occur.  
3. CCM: Pupil-dilation might result in impair vision and increased sensitivity to light for a short 
period of time. Driving is not permitted while vision is impaired. 
4. AF: A transient warm sensation might be felt in relation to the area of medicine application. 
5. Cuff occlusion: There may be discomfort and mild to moderate pain may occur for less than 
5 minutes when inflating the cuff. However, no lasting pain will occur after deflation of the 
cuff.  
6. MRI scanning: There are no known harmful side-effects associated with MRI. However, being 
in the MRI scanner can cause discomfort and claustrophobia. The participant can at any time 
use the emergency squeeze ball to stop the investigation. 
 
C. Benefits to Subjects 
The subjects will not directly benefit from participation. However, the trial might shed some light on a con-
dition, which they either already have or are bound to develop. Furthermore, the findings might help them 
later due to a better understand and prevention/delay of mentioned condition. In addition, the study might 
help their fellow diabetics, as this is a potential new test for early detection and grading of neuropathy, which 
both patients and the scientific community sorely needs. 
 
D. Costs to Subject 
Participants might need to travel to the hospital two additional time(s). No further cost for the participants 
is associated with this project.  
 
E. Compensation to Subject 
No economical compensation will be payed. However, patients may be entitled to a driving allowance in 
accordance to the Danish Health Act. However, neither healthy controls or subjects with diabetes can receive 
driving allowance for visits that are not a part of the standard care (session 1, 2, and 3). In case of unforeseen 
side effects, the patients will be compensated in accordance to the Danish Health Act and “patienterstat-
ningsordningen”. 

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F. Provisions for vulnerable subjects 
The participants are not expected to be any more vulnerable than the average patient is, and the trial does 
not contain any particularly distressing examinations. 
 
X. PLANS FOR DISSEMINATION OF FINDINGS 
The study is to be published as several articles in an internationally acknowledged scientific magazine. The 
results will be published regardless of the outcome of the project and is publish whether accepted in scientific 
journals or not in accordance to “komitelovens §20”. Further dissemination might include conferences like 
Diabetes Foot Study Groups (DFSG) annual meeting. 
 
XI. REFERENCES 
[1] 
P. Hossain, B. Kawar, and M. El Nahas, “Obesity and Diabetes in the Developing World — A Growing 
Challenge,” N. Engl. J. Med., vol. 356, no. 3, pp. 213–215, Jan. 2007. 
[2] 
N. H. Cho et al., “IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and 
projections for 2045,” Diabetes Res. Clin. Pract., vol. 138, pp. 271–281, 2018. 
[3] 
O. H. Y. Yu and S. Suissa, “Identifying causes for excess mortality in patients with diabetes: Closer 
but not there yet,” Diabetes Care, vol. 39, no. 11, pp. 1851–1853, 2016. 
[4] 
A. Bertoni, G. Anderson, J. Kroop, and F. Brancatti, “Diabetes-Related Morbidity and Mortality in a 
National Sample of,” 2002. 
[5] 
A. I. Vinik, M. L. Nevoret, C. Casellini, and H. Parson, “Diabetic Neuropathy,” Endocrinol. Metab. Clin. 
North Am., vol. 42, no. 4, pp. 747–787, 2013. 
[6] 
S. Tesfaye and D. Selvarajah, “Advances in the epidemiology, pathogenesis and management of 
diabetic peripheral neuropathy,” Diabetes. Metab. Res. Rev., vol. 28, no. SUPPL. 1, pp. 8–14, Feb. 
2012. 
[7] 
S. Tesfaye et al., “Painful diabetic peripheral neuropathy: Consensus recommendations on diagnosis, 
assessment and management,” Diabetes. Metab. Res. Rev., vol. 27, no. 7, pp. 629–638, Oct. 2011. 
[8] 
N. K. Chammas, R. L. R. Hill, and M. E. Edmonds, “Increased Mortality in Diabetic Foot Ulcer Patients: 
The Significance of Ulcer Type,” J. Diabetes Res., vol. 2016, 2016. 
[9] 
J. M. Robbins, G. Strauss, D. Aron, J. Long, J. Kuba, and Y. Kaplan, “Mortality rates and diabetic foot 
ulcers: is it time to communicate mortality risk to patients with diabetic foot ulceration?,” J. Am. 
Podiatr. Med. Assoc., vol. 98, no. 6, pp. 489–493, 2008. 
[10] 
F. Crawford, M. Inkster, J. Kleijnen, and T. Fahey, “Predicting foot ulcers in patients with diabetes: a 
systematic review and meta-analysis,” Qjm, vol. 100, no. 2, pp. 65–86, Dec. 2006. 
[11] 
E. J. Boyko, A. D. Seelig, and J. H. Ahroni, “Limb- A nd person-level risk factors for lower-limb 
amputation in the prospective seattle diabetic foot study,” Diabetes Care, vol. 41, no. 4, pp. 891–
898, 2018. 

Perception Threshold Tracking (PTT): A method for early detection and grading of diabetic peripheral neuropathy 
Protocol version: 2 
Side 17 af 23 
 
[12] 
A. A. Fauzi, T. Y. Chung, and L. A. Latif, “Risk factors of diabetic foot Charcot arthropathy: A case-
control study at a Malaysian tertiary care centre,” Singapore Med. J., vol. 57, no. 4, pp. 198–203, 
Apr. 2016. 
[13] 
F. Wang et al., “Diagnostic Accuracy of Monofilament Tests for Detecting Diabetic Peripheral 
Neuropathy: A Systematic Review and Meta-Analysis,” J. Diabetes Res., vol. 2017, p. 8787261, 2017. 
[14] 
A. Tentolouris et al., “The association between pulse wave velocity and peripheral neuropathy in 
patients with type 2 diabetes mellitus,” J. Diabetes Complications, vol. 31, no. 11, pp. 1624–1629, 
2017. 
[15] 
J. Apelqvist, “Diagnostics and treatment of the diabetic foot,” Endocrine, vol. 41, no. 3, pp. 384–397, 
Jun. 2012. 
[16] 
A. J. M. Boulton, “The diabetic foot: Grand overview, epidemiology and pathogenesis,” Diabetes. 
Metab. Res. Rev., vol. 24, no. SUPPL. 1, pp. S3–S6, May 2008. 
[17] 
A. J. M. Boulton, “The pathway to foot ulceration in diabetes,” Med. Clin. North Am., vol. 97, no. 5, 
pp. 775–790, 2013. 
[18] 
J. A. Mayfield, G. E. Reiber, L. J. Sanders, D. Janisse, and L. M. Pogach, “Preventive foot care in 
people with diabetes,” Diabetes Care, vol. 26, no. SUPPL. 1, pp. S78-9, Jan. 2003. 
[19] 
A. Peltier, S. A. Goutman, and B. C. Callaghan, “Painful diabetic neuropathy,” BMJ, vol. 348, no. 
may06 1, pp. g1799–g1799, 2014. 
[20] 
S. Morrison, S. R. Colberg, H. K. Parson, and A. I. Vinik, “Relation between risk of falling and postural 
sway complexity in diabetes,” Gait Posture, vol. 35, no. 4, pp. 662–668, 2012. 
[21] 
S. Tesfaye et al., “Prevalence of diabetic peripheral neuropathy and its relation to glycaemic control 
and potential risk factors: The EURODIAB IDDM Complications Study,” Diabetologia, vol. 39, no. 11, 
pp. 1377–1384, 1996. 
[22] 
R. E. Maser et al., “Epidemiological correlates of diabetic neuropathy. Report from Pittsburgh 
epidemiology of diabetes complications study,” Diabetes, vol. 38, no. 11, pp. 1456–1461, Nov. 1989. 
[23] 
C. L. Martin, J. W. Albers, and R. Pop-Busui, “Neuropathy and related findings in the diabetes control 
and complications trial/epidemiology of diabetes interventions and complications study,” Diabetes 
Care, vol. 37, no. 1, pp. 31–38, 2014. 
[24] 
S. Tesfaye et al., “Diabetic neuropathies: Update on definitions, diagnostic criteria, estimation of 
severity, and treatments,” Diabetes Care, vol. 33, no. 10, pp. 2285–2293, Oct. 2010. 
[25] 
N. Chammas, R. Hill, A. Foster, and M. Edmonds, “Is Neuropathic Ulceration the Key to 
Understanding Increased Mortality Due to Ischaemic Heart Disease in Diabetic Foot Ulcer Patients? 
A Population Approach Using a Proportionate Model,” 2002. 
[26] 
J. Van Baal, R. Hubbard, F. Game, and W. Jeffcoate, “Mortality associated with acute charcot foot 
and neuropathic foot ulceration,” Diabetes Care, vol. 33, no. 5, pp. 1086–1089, May 2010. 
[27] 
Sanjay Kalra Anu Gupta, “Diabetic Painful Neuropathy and Restless Legs Syndrome in Diabetes,” 
Diabetes Ther., vol. 9, no. 2, pp. 441–447, 2018. 
[28] 
G. J. Biessels et al., “Phenotyping animal models of diabetic neuropathy: A consensus statement of 
the diabetic neuropathy study group of the EASD (Neurodiab),” J. Peripher. Nerv. Syst., vol. 19, no. 2, 
pp. 77–87, Jun. 2014. 

Perception Threshold Tracking (PTT): A method for early detection and grading of diabetic peripheral neuropathy 
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[29] 
P. D. O’Brien, L. M. Hinder, S. A. Sakowski, and E. L. Feldman, “ER Stress in Diabetic Peripheral 
Neuropathy: A New Therapeutic Target,” Antioxid. Redox Signal., vol. 21, no. 4, pp. 621–633, 2014. 
[30] 
A. M. Vincent, B. C. Callaghan, A. L. Smith, and E. L. Feldman, “Diabetic neuropathy: Cellular 
mechanisms as therapeutic targets,” Nat. Rev. Neurol., vol. 7, no. 10, pp. 573–583, 2011. 
[31] 
R. Pop-Busui et al., “Diabetic neuropathy: A position statement by the American diabetes 
association,” Diabetes Care, vol. 40, no. 1, pp. 136–154, 2017. 
[32] 
R. A. Malik et al., “Sural nerve pathology in diabetic patients with minimal but progressive 
neuropathy,” Diabetologia, vol. 48, no. 3, pp. 578–585, 2005. 
[33] 
A. G. Smith, P. Ramachandran, S. Tripp, and J. R. Singleton, “Epidermal nerve innervation in,” 
Neurology, 2010. 
[34] 
S. Yagihashi, S. I. Yamagishi, and R. Wada, “Pathology and pathogenetic mechanisms of diabetic 
neuropathy: Correlation with clinical signs and symptoms,” Diabetes Res. Clin. Pract., vol. 77, no. 3 
SUPPL., 2007. 
[35] 
G. F. Ziegler, D, Mayer P, Mühlen H, “The natural history of somatosensory and autonomic nerve 
dysfunction in relation to glycaemic control during the first 5 years after diagnosis of type 1 (insulin-
dependent) diabetes mellitus,” Diabetologia, pp. 822–829, 1991. 
[36] 
J. Dros, A. Wewerinke, P. J. Bindels, and H. C. Van Weert, “Accuracy of monofilament testing to 
diagnose peripheral neuropathy: A systematic review,” Ann. Fam. Med., vol. 7, no. 6, pp. 555–558, 
Nov. 2009. 
[37] 
L. S. Tan, “The clinical use of the 10g monofilament and its limitations: A review,” Diabetes Res. Clin. 
Pract., vol. 90, no. 1, pp. 1–7, 2010. 
[38] 
G. Ponirakis et al., “The diagnostic accuracy of Neuropad for assessing large and small fibre diabetic 
neuropathy.,” Diabet. Med., vol. 31, no. 12, pp. 1673–80, Dec. 2014. 
[39] 
N. Papanas and D. Ziegler, “New diagnostic tests for diabetic distal symmetric polyneuropathy ☆,” J. 
Diabetes Complications, vol. 25, pp. 44–51, 2010. 
[40] 
D. Ziegler, E. Siekierka-Kleiser, B. Meyer, and M. Schweers, “Validation of a novel screening device 
(NeuroQuick) for quantitative assessment of small nerve fiber dysfunction as an early feature of 
diabetic polyneuropathy,” 2005. 
[41] 
F. L. Bowling, C. A. Abbott, W. E. Harris, S. Atanasov, R. A. Malik, and A. J. M. Boulton, “A pocket-
sized disposable device for testing the integrity of sensation in the outpatient setting,” Diabet. Med., 
vol. 29, no. 12, pp. 1550–1552, 2012. 
[42] 
A. I. Vinik, X. Kong, J. T. Megerian, and S. N. Gozani, “Diabetic nerve conduction abnormalities in the 
primary care setting.,” 2006. 
[43] 
J. H. Calvet, J. Dupin, H. Winiecki, and P. E. H. Schwarz, “Assessment of small fiber neuropathy 
through a quick, simple and non invasive method in a German diabetes outpatient clinic,” Exp. Clin. 
Endocrinol. Diabetes, vol. 121, no. 2, pp. 80–83, 2013. 
[44] 
G. Lauria et al., “Intraepidermal nerve fiber density at the distal leg: A worldwide normative 
reference study,” J. Peripher. Nerv. Syst., vol. 15, no. 3, pp. 202–207, 2010. 
[45] 
C. Quattrini et al., “Surrogate markers of small fiber damage in human diabetic neuropathy,” 
Diabetes, vol. 56, no. 8, pp. 2148–2154, 2007. 

Perception Threshold Tracking (PTT): A method for early detection and grading of diabetic peripheral neuropathy 
Protocol version: 2 
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[46] 
N. Papanas and D. Ziegler, “Corneal confocal microscopy: A new technique for early detection of 
diabetic neuropathy,” Curr. Diab. Rep., vol. 13, no. 4, pp. 488–499, Aug. 2013. 
[47] 
L. Arendt-Nielsen and D. Yarnitsky, “Experimental and Clinical Applications of Quantitative Sensory 
Testing Applied to Skin, Muscles and Viscera,” J. Pain, vol. 10, no. 6, pp. 556–572, 2009. 
[48] 
E. K. Krumova, C. Geber, A. Westermann, and C. Maier, “Neuropathic pain: Is quantitative sensory 
testing helpful?,” Curr. Diab. Rep., vol. 12, no. 4, pp. 393–402, 2012. 
[49] 
N. Papanas et al., “Use of the new indicator test (Neuropad®) for the assessment of the staged 
severity of neuropathy in type 2 diabetic patients,” Exp. Clin. Endocrinol. Diabetes, vol. 115, no. 1, 
pp. 58–61, 2007. 
[50] 
N. Papanas and D. Ziegler, “New vistas in the diagnosis of diabetic polyneuropathy,” Endocrine, vol. 
47, no. 3, pp. 690–698, Dec. 2014. 
[51] 
S. Sharma, C. Kerry, H. Atkins, and G. Rayman, “The Ipswich Touch Test: A simple and novel method 
to screen patients with diabetes at home for increased risk of foot ulceration,” Diabet. Med., vol. 31, 
no. 9, pp. 1100–1103, 2014. 
[52] 
B. A. Perkins, J. Grewal, E. Ng, M. Ngo, and V. Bril, “Validation of a novel point-of-care nerve 
conduction device for the detection of diabetic sensorimotor polyneuropathy,” Diabetes Care, vol. 
29, no. 9, pp. 2023–2027, 2006. 
[53] 
L. Vileikyte, G. Hutchings, S. Hollis, and A. J. M. Boulton, “The tactile circumferential discriminator: A 
new, simple screening device to identify diabetic patients at risk of foot ulceration,” Diabetes Care, 
vol. 20, no. 4, pp. 623–626, Apr. 1997. 
[54] 
N. Papanas, A. Gries, E. Maltezos, and R. Zick, “The steel ball-bearing test: A new test for evaluating 
protective sensation in the diabetic foot,” Diabetologia, vol. 49, no. 4, pp. 739–743, Apr. 2006. 
[55] 
H. Gin, R. Baudoin, C. H. Raffaitin, V. Rigalleau, and C. Gonzalez, “Non-invasive and quantitative 
assessment of sudomotor function for peripheral diabetic neuropathy evaluation,” Diabetes Metab., 
vol. 37, no. 6, pp. 527–532, 2011. 
[56] 
C. M. Casellini, H. K. Parson, M. S. Richardson, M. L. Nevoret, and A. I. Vinik, “Sudoscan, a 
Noninvasive Tool for Detecting Diabetic Small Fiber Neuropathy and Autonomic Dysfunction,” 
Diabetes Technol. Ther., vol. 15, no. 11, pp. 948–953, 2013. 
[57] 
J. M. Slade, T. F. Towse, V. V. Gossain, and R. A. Meyer, “Peripheral microvascular response to 
muscle contraction is unaltered by early diabetes but decreases with age,” J. Appl. Physiol., vol. 111, 
no. 5, pp. 1361–1371, 2011. 
[58] 
M. Vaeggemose et al., “Magnetic resonance neurography visualizes abnormalities in sciatic and 
tibial nerves in patients with type 1 diabetes and neuropathy,” Diabetes, vol. 66, no. 7, pp. 1779–
1788, 2017. 
[59] 
M. N. Nouri et al., “Diabetic neuropathy and axon reflex-mediated neurogenic vasodilatation in type 
1 diabetes,” PLoS One, vol. 7, no. 4, p. 34807, 2012. 
[60] 
M. L. Kubasch et al., “Laser Doppler assessment of vasomotor axon reflex responsiveness to 
evaluate neurovascular function,” Front. Neurol., vol. 8, no. AUG, p. 370, 2017. 
[61] 
D. Fuchs, P. P. Dupon, L. A. Schaap, and R. Draijer, “The association between diabetes and dermal 
microvascular dysfunction non-invasively assessed by laser Doppler with local thermal hyperemia: A 
systematic review with meta-analysis,” Cardiovasc. Diabetol., vol. 16, no. 1, p. 11, 2017. 

Perception Threshold Tracking (PTT): A method for early detection and grading of diabetic peripheral neuropathy 
Protocol version: 2 
Side 20 af 23 
 
[62] 
K. Hennings, K. S. Frahm, L. Petrini, O. K. Andersen, L. Arendt-Nielsen, and C. D. Mørch, “Membrane 
properties in small cutaneous nerve fibers in humans,” Muscle Nerve, vol. 55, no. 2, pp. 195–201, 
2017. 
[63] 
F. Becker et al., “Chapter I: Definitions, epidemiology, clinical presentation and prognosis,” 2011. 
[64] 
R. Freynhagen, R. Baron, U. Gockel, and T. R. Tölle, “pain DETECT : a new screening questionnaire to 
identify neuropathic components in patients with back pain,” Curr. Med. Res. Opin., vol. 22, no. 10, 
pp. 1911–1920, 2006. 
[65] 
A. Jespersen et al., “Is neuropathic pain underdiagnosed in musculoskeletal pain conditions? The 
Danish PainDETECTive study,” Curr. Med. Res. Opin., vol. 26, no. 8, pp. 2041–2045, 2010. 
[66] 
R. Rolke et al., “Quantitative sensory testing: A comprehensive protocol for clinical trials,” Eur. J. 
Pain, vol. 10, no. 1, pp. 77–88, 2006. 
[67] 
H. H. Andersen, J. Elberling, S. Lo Vecchio, and L. Arendt-Nielsen, “Topography of itch,” Itch, vol. 2, 
no. 1, p. e2, 2017. 
[68] 
H. Bostock, K. Cikurel, and D. Burke, “Threshold tracking techniques in the study of human 
peripheral nerve.,” Muscle Nerve, vol. 21, no. 2, pp. 137–158, 1998. 
 

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XII. APPENDICES   
PainDETECT 
 
 

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Consent form 
See separate document. 
 
Flowchart 
Sessions mentioned below can be conducted in any order as long as the sessions are completed with 3 
months.