Investigation of the Relationship Between Gremlin-1 Levels and Wagner Classification inDiabetic Foot Patients and Evaluation of Its Usability as a Biomarker in the Progress toAmputation
Conditions: Diabetes Mellitus, Diabetic Foot Disease, Diabetic Foot Ulcer, Diabetic Neuropathy, Peripheral Arterial Disease
Sponsor: Seyma Acik

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Gremlin-1 and Wagner Classification: Potential Biomarker 
for Amputation in Diabetic Foot Patients  
 
STUDY PROTOCOL 
This study is a cross-sectional, single-center study conducted between 20xx and 20xx. A total of 85 
patients aged 18 years and older, diagnosed with DM, and who either had diabetic foot ulcers or were 
at high risk for DF, were recruited from the Endocrinology outpatient clinic or assessed by the diabetic 
foot council of XXX University Faculty of Medicine Hospital. All patients agreed to participate in the 
study and to share their data in accordance with the principles of the Helsinki Declaration (2001). 
Approval from the XXX University Clinical Research Ethics Committee was obtained on December 
XX, XXXX (approval number XXX). 
Patients with autoimmune diseases, malignant conditions, a history of prior amputation, those on 
hemodialysis, and pregnant women were excluded from the study. 
Data were collected on patient age, smoking status, type of diabetes, duration of diabetes, treatments 
received, and diabetes-related complications. 
Hematocrit, C-reactive protein (CRP), white blood cell (WBC) count, platelets, HbA1c, triglycerides, 
cholesterol levels, glomerular filtration rate (GFR), and albuminuria data were extracted using electronic 
medical records. 
Diabetic peripheral neuropathy was evaluated based on the patient's medical history, clinical assessment, 
and diagnostic tests. The Semmes-Weinstein monofilament test was used to assess diabetic peripheral 
neuropathy. 
Retinopathy was defined based on ophthalmological evaluations as the presence of non-proliferative or 
proliferative diabetic retinopathy. 
Cardiovascular disease (CVD) was considered positive if the patient had a history of bypass surgery, 
stent placement, positive angiography, or myocardial infarction. Patients with normal angiography and 
exercise test results or without a history of coronary artery disease were classified as CVD negative. 
Albuminuria was defined as an albumin-to-creatinine ratio >30 mg/g during a spot urine test. 

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DF severity was classified using the Wagner diabetic foot classification system, based on clinical and 
imaging assessments after identifying the ulcer site and presence of infection. Patients with DF ulcers 
in stages 1 and 2 (limited to tissue) were evaluated together with patients in stages 4 and 5 (with 
necrosis). 
The Ankle Brachial Index (ABI) of the patients was measured using both the ankle and both arms after 
a 30-minute rest period.  
Symptomatic peripheral artery disease (PAD) was considered present if the ABI was <0.9, if imaging 
was consistent with PAD, or if the patient had a history of stent placement or revascularization. Contrast-
enhanced extremity imaging techniques included computed tomography angiography, magnetic 
resonance angiography, or Doppler ultrasonography. ABI values between 0.9-1.3 were considered 
normal, ABI <0.9 was classified as PAD, and values >1.3 (calcified) were excluded. 
In addition to routine tests, a 5 mL blood sample was drawn from the antecubital veins of all patients 
for the measurement of serum Gremlin-1 levels. The blood samples were sent to the Department of 
Medical Biochemistry at XXX University Faculty of Medicine. Blood collected in serum tubes was 
centrifuged at 4000 rpm for 5 minutes, and the serum was separated. The serum samples were stored at 
-80°C in 1.5 mL Eppendorf tubes until the day of analysis. On the day of analysis, the serum samples 
were brought to room temperature, and Gremlin-1 protein levels were analyzed using the ELISA method 
(Novus) following the procedure. The Gremlin-1 protein levels were evaluated in ng/mL. 
 
 

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Gremlin-1 and Wagner Classification: Potential Biomarker 
for Amputation in Diabetic Foot Patients  
STATISTICAL ANALYSIS PLAN 
In the statistical analysis of the data, categorical variables were examined using frequency tables, and 
descriptive statistics for continuous variables were calculated. Pearson’s chi-square test was used for 
categorical variables, and the levels of the variables were analyzed using cross-tabulations. The Shapiro-
Wilk normality test was used to determine whether continuous variables were normally distributed. 
Since the assumption of normal distribution was not met, the Mann-Whitney U test was used to compare 
two independent groups. For the comparison of non-normally distributed variables among more than 
two groups, the Kruskal-Wallis test was used. Correlations between continuous variables were examined 
using the Spearman correlation coefficient. A significance level of 0.05 was applied to all group 
comparisons. IBM SPSS Version 25.0 statistical software package was used for the statistical analysis. 
 
 
 

                                                                                                                                                                  20.12.2019 
Gremlin-1 and Wagner Classification: Potential Biomarker 
for Amputation in Diabetic Foot Patients  
 
INFORMED CONSENT FORM 
PLEASE READ CAREFULLY!!! 
 
You have been invited to participate in this study. Before accepting to take part in this study, you 
must understand the purpose of the study and make your decision freely after this information. Please 
read this information prepared specifically for you carefully and ask for clear answers to your 
questions. 
What is the name of this study? 
 
Investigation of the Relationship between Gremlin-1 Levels and Wagner Classification in Diabetic 
Foot Patients and Testing Its Usability as a Biomarker for Amputation 
What is the purpose of this study? 
 
In patients with diabetic foot wounds, gremlin-1 levels will be studied in the blood to predict the 
degree of the wound, to determine the course of amputation (the surgical removal of an organ that is 
deemed impossible to heal), and to determine the treatment method for this. If it is significant, it will 
be in the interest of patients to determine the severity of the disease with a blood test and to apply 
early treatment methods. 
How will you be treated? 
 
Your routine examinations and tests will not be affected. For this study, we will only need to take a 5 
ml blood sample from the vein in your arm for testing. The blood collection procedure will be 
performed by a doctor or healthcare personnel in accordance with the blood collection procedure. 
What is the probability of being randomly assigned to different treatment groups? 

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Our study is not testing any treatment method. There will be no changes to your current treatment. 
How much time will it take? 
Aside from your routine examination, 5-10 minutes will be enough time for us to take a blood sample. 
What is the estimated number of volunteers expected to participate in the study? 
 
90 
What will happen to the biological materials that will be taken from you, and where will the 
analyses be done? (explanation of where the biological materials will be sent if the analyses are 
done abroad), 
 
5 ml blood samples taken from the participants will be examined in the Biochemistry Laboratory of 
the Ege University Faculty of Medicine and then destroyed. 
What is expected of you? What are your responsibilities? 
 
It will be sufficient to provide a 5 ml blood sample. 
What will you benefit from participating in the study? 
 
A method that is likely to be a guide in the follow-up and treatment of your disease will be tested. 
The results are expected to provide innovation/contribution to the future follow-up and treatment of 
you and other patients. 
What are the situations that would require termination of participation in the study? 
 
You may withdraw from the study at any time if you withdraw your written consent. 
Could participating in the study cause you any harm? 
No 

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What happens if you do not want to participate? 
 
There will be no change for you. Your follow-up and treatment will continue as is. 
What alternative methods can be applied to you? 
There is no alternative method for the study. 
Will I be paid anything for participating in this study? 
 
No 
Will I pay any fees for participating in this study? 
 
All kinds of tests, physical examinations, and other research expenses will not be paid by you or any 
official or private institution or organization that you are under the guarantee of. 
Confidentiality of information: All your personal and medical information will remain 
confidential and will only be used for scientific purposes. Even if the research results are 
published, your identity will remain confidential. 
Contact information of the person responsible for this study 
1- Name, surname: Prof. Dr. xxxxxxx 
2-  Reachable phone number: xxxxxxX 
3- Position: XXX University Department of Internal Medicine, Department of 
Endocrinology 
 
 
Consent to Participate in the Study 
 
I have read and listened to the information provided above, which indicates that the subject should be 
given before the study begins. I have asked the researcher all questions that come to mind and have 
fully understood all the explanations given to me in writing and verbally. I have been given sufficient 

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time to decide whether I wish to participate in the study. Under these conditions, I authorize the 
researcher to review, transfer and process my medical information and I accept the invitation to 
participate in this study with great willingness, without any coercion or pressure. I understand that 
my participation in the study is voluntary and that I may withdraw from the study with or without 
giving any reason. I understand that by signing this form I will not lose any rights granted to me by 
local law. 
 
I understand that I will be given a signed and dated copy of the informed consent. 
 
Please select one of the following options: 
 
Biological samples taken within the scope of the research titled 'Investigation of the Relationship 
Between Gremlin-1 Levels and Wagner Classification in Diabetic Foot Patients and Testing Its 
Usability as a Biomarker for Amputation' 
     -I only allow it to be used in the above-mentioned research. 
  -I allow it to be used in future research limited to the relevant disease group. 
  -I do not allow it to be used under any circumstances. 
. 
 
 
VOLUNTEER'S 
SIGN 
NAME-
SURNAME 
 
 
ADRESS 
 
TELEPHONE 
 
DATE 
 

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For those under guardianship or trusteeship, the parent or guardian 
SIGN 
NAME-
SURNAME 
 
 
ADRESS 
 
TELEPHONE 
 
DATE 
 
 
A competent researcher who is a part of the research team and provides information 
about the research. 
SIGN 
NAME-
SURNAME 
 
 
ADRESS 
 
TELEPHONE 
 
DATE