Comparative Diagnostic Accuracy Study of Bedside Tests Used to Detect Arterial Disease in Diabetes:
Conditions: Diabetic Foot, Peripheral Arterial Disease, Diabetic Foot Ulcer, Peripheral Vascular Diseases, Peripheral Artery Disease, Critical Limb Ischemia, Critical Lower Limb Ischemia
Sponsor: Imperial College Healthcare NHS Trust

    Study Protocol and Statistical Analysis Plan 
 
Study title: TEsting for Arterial Disease in Diabetes (TrEAD) Study 
 
NCT number: NCT04058626 
 
Date: 01/02/2019 
 

 
INTRODUCTION 
 
Diabetic foot complications affect up to  25% of people with diabetes at some 
point in their life are associated with high rates of amputation and mortality 
(1,2). Ulcers are also challenging to manage, necessitating intensive treatment 
which costs the National Health Service (NHS) an estimated £1 billion per year 
(3).  However, it has been suggested that up to 80% of diabetic amputations are 
preventable (4) and that meaningful impact can still be made.  
 
Neuropathy and peripheral arterial disease (PAD) are the most important risk 
factors for the development of foot ulceration (5). With rising prevalence of PAD 
in western populations, there has been a shift of pattern from neuropathic to 
‘neuro-ischaemic’ ulceration (6–8). The prompt and accurate detection of PAD in 
the ulcerated foot is time critical as time to revascularisation is a determinant of 
ulcer healing and limb salvage (9,10). This serves as an important target for 
improving ulcer care due to the confounding effects of neuropathy and arterial 
calcification, the detection of PAD in diabetes is challenging. Symptoms, such as 
claudication and rest pain are often masked by the presence of neuropathy. 
Furthermore, physical examination and bedside screening tests such as ankle 
brachial pressure indices (ABPI) are hindered by the presence of calcified, 
incompressible blood vessels. Limited evidence suggests that toe brachial 
pressure indices (TBPI) and transcutaneous pressure of oxygen (TcPO2) may be 
superior to ABPI measurements (11–13). However, TBPI may also be influenced 
by the presence of digital artery calcification and TcPO2 can be time consuming 
to perform and may not be feasible in real world diabetic foot practice, 
reportedly used 6.2 and 0.8% respectively (14). Interpretation of audible 
Doppler arterial signals using hand held Doppler devices is the most commonly 
used bedside test, but this is also limited by significant inter-observer variation 

and poor sensitivity in detecting PAD (15,16). Currently, there is a scarcity of 
robust evidence to support the use of any one test (11).  
 
The limitations of current bedside tests highlight a need for a more accurate and 
reliable non-invasive screening tool for the detection of arterial disease. The lack 
of evidence highlights the need for a comparative diagnostic accuracy study to 
identify the best screening strategy. 
 
Point-of-care arterial duplex ultrasound may provide an alternative solution to 
diagnosis. A full lower limb arterial DUS scan has an accuracy comparable to the 
gold standard of angiography (17) but is too time consuming to perform and 
technically challenging to learn due to the difficulty of scanning proximal tibial 
vessels (18), thus limiting its utility as a bed-side test by front line staff. Focused 
scanning of the anterior and posterior tibial arteries at the ankle can provide 
information regarding the upstream state of the vasculature. We have given this 
focused DUS the acronym PAD-scan (Podiatry Ankle Duplex Scan). The PAD-scan 
allows for direct vessel visualisation and selective sampling of the Doppler signal 
from a region of interest to detect arterial disease. This reduces the chance of 
mistakenly sampling collaterals and allows for more granular waveform analysis 
by sampling from a region of the vessel with low calcification. We have 
previously demonstrated that the PAD-scan can be readily learned and 
accurately performed by front line staff (19).   
 
The primary aim of this present study is to determine the ability of the PAD-scan, 
continuous handheld Doppler (audible and visual waveform assessment), ABPI, 
TBPI, TcPO2 and pulse palpation to discriminate between the presence and 
absence of PAD in diabetes, as compared to a reference standard of full lower 
limb arterial DUS. 
 
The results of this study will improve the diagnosis of PAD in diabetes and 
inform referral pathways between primary and secondary care.  Early detection, 
referral and management of PAD in the ulcerated foot will result in faster healing 
rates, reduction in hospital admissions, reduction in lower limb amputations and 

cost savings for the NHS. In the non-ulcerated foot, detection of PAD may alter 
foot care behaviours to reduce the risk of ulceration and allow for optimisation 
of best medical therapy to reduce overall cardiovascular risk.   
METHODS AND ANALYSIS 
 
Centre(s): St Mary’s Hospital (London, UK), West Middlesex University Hospital 
(London, UK). 
 
Design 
Inclusion and exclusion criteria:  
Diabetic patients aged over 18 years presenting to the diabetic foot clinics at our 
chosen Centre’s will be eligible for the study. Patients with a known history of 
vascular imaging or intervention in the past 1 year will be excluded. Patients 
unable to provide informed consent will also be excluded. 
 
Intervention(s) or method: 
 
Patient assessment: 
The clinical team responsible for care will perform a routine clinical assessment 
of each patient. Neuropathy will be tested for using a 10-g monofilament, 128Hz 
tuning fork and proprioception at the first metatarsophalangeal joint. 
Assessment of ulcer size, depth and severity will be made using the validated 
University of Texas (UT) Score (20,21).  
 
Index tests: 
Pulse palpation and audible handheld continuous Doppler assessment will be 
performed by the Podiatrist assessing the patient as part of their routine clinical 
assessment. 
This present study focuses on diagnostic accuracy and not feasibility of frontline 
staff learning or performing the PAD-scan, which we have previously 
established. Therefore, two vascular scientists will perform all tests, one of the 

two will perform the index tests; PAD-scan, TcPO2, ABPI, TBPI and visual 
handheld continuous Doppler waveform assessment, the other will perform the 
reference full Duplex scan. Each scientist will be blinded to the findings of the 
other.  
 
• Pulse palpation 
The dorsalis pedis (DPA) and posterior tibial artery (PTA) pulses will be 
palpated and classified as present or absent. Absence of either pulse will be 
considered diagnostic. 
 
• Continous handheld Doppler device (audible and visual waveform 
assessment) 
The DPA and PTA will be interrogated using a continuous wave handheld 
Doppler device. The presence of a monophasic signal or the absence of a signal in 
either vessel will be considered diagnostic. Secondary analysis will also consider 
the inclusion of biphasic waveforms to the diagnostic criteria.  
 
• ABPI 
A sphygmomanometer-cuff placed at the ankle and a handheld continuous wave 
Doppler device will be used to measure the systolic pressure of the DPA and PTA. 
The pressures of both arms will also be taken and the highest reading used to 
calculate the ABPI (22). ABPI of ≤ 0.9 will be considered diagnostic. Secondary 
analysis will consider the inclusion of ABPI >1.3 (23) to the diagnostic criteria.   
 
• PAD-scan 
Podiatry ankle duplex scan (PAD-scan) involves using an ultrasound machine to 
visualise the anterior and posterior tibial arteries at the ankle, which supply the 
foot. Graphically displayed Doppler spectral waveforms are then assessed. PAD-
scan is positive for the presence PAD in the presence of an occlusion, venous like 
slow flow, monophasic waveform, or a biphasic waveform with adverse features 
(slow rise time, spectral broadening, infilling of the spectral window and long 
forward flow in diastole) in either vessel scanned. All scans will be performed 

using the Mindray M7 (Shenzhen, China) Portable Ultrasound System with a 
linear 6–14 kHz transducer.  
 
• TBPI 
 
TBPI will be measured using the photoplethysmography (PPG) method, 
employing an infrared sensor placed on the hallux and index finger. The highest 
upper limb reading will be used to calculate the TBPI. Values of <0.75 will be 
considered diagnostic for PAD (24).  
 
• TcPO2 
Prior to taking TcPO2 measurements, patients will be acclimatized for 20 
minutes in the resting supine position with the room temperature maintained 
between 23 and 25 °C.  Calibration will be performed and transducers placed on 
the skin after cleaning using double-sided adhesive rings and contact liquid (25). 
Measurements will be taken centrally (at the sternum or the deltoid if the patient 
has had a previous midline sternotomy) and on the dorsum of the foot using an 
automated machine equipped with Clark electrodes. Foot measurements will be 
repeated after 3 minutes of 30° leg elevation supported by a wedge. Readings of 
<40mmHg at resting supine position in the foot electrode will be considered 
diagnostic (26). Regional Perfusion Index (RPI, limb TcPO2 values normalised to 
central values; values <0.6 considered diagnostic) and change in TcPO2 on leg 
elevation (decrease of >10mmHg considered diagnostic) will also be calculated 
as secondary TcPO2 metrics of interest for PAD diagnosis.  
 
Reference test: 
Intra-arterial digital subtraction angiography (DSA) has long been considered 
the gold standard for diagnosis of PAD. However, it is expensive, invasive and 
carriers a risk of complications. DUS has been shown to be comparable to DSA, 
making it a useful non-invasive alternative (17). 
 
Patients will undergo a full departmental DUS at their respective hospitals on the 
same day as the index tests by one of two blinded vascular scientists present in 
each clinic. PAD will be deemed significant when occlusions, or stenosis, or 

diffuse stenotic disease, individually or collectively, cause significant velocity 
change (peak systolic velocity ratio (PSVR) ³2) and flow disturbance locally and 
result in biphasic or monophasic signal distally. All scans will be performed using 
the Mindray M7 (Shenzhen, China) Portable Ultrasound System with a linear 6–
14 kHz transducer. 
 
Quality assurance: 
PAD-scan: For quality assurance, an external vascular scientist will review a 
random selection of 40 saved scans. 
Reference test: Quality will be assured by acquiring a second DUS by an external 
vascular scientist in 20 individuals.  
Statistical analysis and plan: 
 
Sample size and power calculations: 
Calculations were performed using the described sample size formula for 
estimating sensitivity (27). Calculations were based on a 95% confidence level 
(i.e. an error of 5%), an estimated PAD prevalence of 50% (28) and a predicted 
sensitivity of 90% for the PAD-scan as derived from our previous research. The 
estimated sample size required was calculated as 277 patients. From previous 
research undertaken, we anticipate an attrition rate of 10%. Therefore, the 
recruitment target for this study is 305 patients. 
 
Statistical tests:   
Cross-tabulation of the index test results by the results of the reference standard 
will be made creating a ‘confusion matrix’. This will be used to determine 
diagnostic accuracy, expressed as sensitivity, specificity, predictive values and 
likelihood ratios. Receiver operating characteristic (ROC) analysis will also be 
performed for each test. Agreement between tests will be analysed using the 
Cohen’s kappa statistic. Statistical modeling will also be performed to explore the 
effect of combining tests on diagnostic accuracy in order to identify the most 
accurate diagnostic strategy. 

Ethics 
Ethics Committee approval: NHS Health Research Authority (NRES Committee 
London- City & East Research Ethics Committee) approval gained on 
21/12/2017 (REC reference 17/LO/1447).  
 
Interim analysis and stopping rules: No interim analysis will be performed, 
and the data will be analysed when the target recruitment of 305 patients has 
been reached.  
PATIENT AND PUBLIC INVOLVEMENT 
 
Patient representatives were involved in the design of this project protocol. In 
particular they informed the recruitment process in order to ensure that it was 
acceptable to patients. After completion of the study they will also be invited to a 
roundtable discussion of our findings, allowing for integration of their 
perspectives with those of practitioners. They will also be involved in the 
dissemination of our findings via patient groups and the production of written 
and video summaries for patients.  
DISSEMINATION 
The results of the study will be published a peer-reviewed journal and presented 
at international surgical and diabetes research meetings. Furthermore, to ensure 
that our findings reach all stakeholders including patients our results will also be 
presented at relevant patient group meetings as well as local and regional 
diabetic foot network meetings.    
 
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