Diagnostic Accuracy of Cold, Heat, and Electric Pulp Tests: An In Vivo Study
Conditions: Dental Pulp Disease, Pulp Necrosis, Pulpitis - Irreversible
Sponsor: Istanbul University

 
 
Diagnostic Accuracy of Cold, Heat, and Electrical Pulp Tests: 
An In Vivo Study 
Short Title: Diagnostic Accuracy of Pulp Sensibility Tests 
Institution / Sponsor 
Istanbul University Faculty of Dentistry 
Study Site 
Department of Endodontics, Istanbul University Faculty of 
Dentistry 
Istanbul, Türkiye 
Ethics Approval 
Ethical Committee Ref. 2024/48 Rev-2 (05.06.2024) 
TİTCK Approval 
Protocol No. 2023/38 
ClinicalTrials.gov ID 
NCT Number: [Not yet assigned] 
 
Document Date: 05.06.2024 
 
 
 

Study Protocol 
Background and Rationale 
Accurate assessment of pulp tissue status is essential for endodontic diagnosis and 
appropriate treatment planning. Pulp sensibility tests -including cold, heat, and electric pulp 
testing- are widely used in routine clinical practice; however, their performance may vary 
across clinical conditions and patient populations. Direct assessment of pulpal bleeding during 
access cavity preparation is commonly considered a practical reference for pulp vitality in 
clinical settings (1-7). This study evaluates the diagnostic performance of commonly used pulp 
sensibility tests against pulpal bleeding as the reference standard in an endodontic clinical 
population. 
Study Objectives 
Primary Objective 
To evaluate the predictive performance of cold, heat, and electric pulp sensibility tests for 
determining pulp vitality, using pulpal bleeding during access cavity preparation as the 
reference standard. 
Secondary Objectives 
• 
To quantify false-positive and false-negative responses for each test relative to the 
reference standard. 
• 
To estimate sensitivity, specificity, and overall accuracy for each test. 
• 
To assess agreement between each index test and the reference standard using Cohen’s 
kappa. 
• 
To compare test results across cold, heat, and EPT using appropriate paired statistical 
methods. 
Study Design 
Prospective, observational, in vivo diagnostic accuracy study conducted in routine care at a 
university endodontics clinic. Participants are not assigned to interventions; index tests are 
applied as standardized diagnostic procedures. 
Study Population and Setting 
Participants are selected from patients presenting to the Endodontics Clinic at Istanbul 
University Faculty of Dentistry, including patients referred from the Department of Oral and 
Maxillofacial Radiology, who are evaluated in routine care and determined to require 
endodontic treatment for at least one tooth. 
 
 

Eligibility Criteria 
Inclusion Criteria 
• 
Age ≥18 years 
• 
At least one tooth indicated for root canal treatment (primary treatment or 
nonsurgical retreatment), confirmed at the initial examination unit 
• 
Able and willing to provide written informed consent 
• 
One tooth per participant included 
Exclusion Criteria 
Tooth-related: full-coverage crowns, extensive restorations affecting testing area, history of 
trauma (study tooth), root resorption, incomplete root development, fractures/cracks, 
regressed 
pulp 
chambers 
or 
calcified 
root 
canals. 
Patient-related: severe or uncontrolled systemic disease (ASA physical status 3–6). 
Sample Size 
Sample size was determined using the mean prevalence reported in comparable diagnostic 
accuracy studies of pulp sensibility tests (assumed prevalence: 45%) (2, 4, 5, 8-11) . With 95% 
confidence, expected sensitivity/specificity of 95%, and a 5% margin of error, the minimum 
required sample was 163 teeth. To allow for potential exclusions and to improve precision, a 
total of 175 participant were included in the study, with one tooth per participant selected. 
Recruitment and Consent 
Eligible participants were identified during routine clinical evaluation. According to the ethical 
principles of the Declaration of Helsinki, informed and written consent were obtained from 
the patients before the clinical tests. 
Blinding and Roles 
Index test operator: performs pulp sensibility tests; blinded to clinical signs/symptoms, dental 
history, and radiographic findings; and blinded to any prior sensibility test results obtained 
during routine evaluation. 
Reference standard assessor: prepares the acces cavity and asseses pulpal bleeding; blinded 
to index test results. 
Assessors are blinded to each other’s findings. 
Test Standardization 
• 
Teeth were isolated with cotton rolls and dried with an air syringe. 
• 
Participants were intructed to raise their hand immediately upon perceiving pain, 
discomfort or tingling. 

• 
Baseline response was obtained from an asymptomatic, functional contralateral tooth 
without prior endodontic treatment (or an adjacent tooth if contralateral is 
unavailable). The test tooth was tested last. 
• 
Pulp tests were applied to standardized areas: incisors/canines (middle third buccal), 
premolars (occlusal third), molars (mesiobuccal cusp tip area or closest available site). 
Pulp Sensibility Tests 
Order of tests: Cold → Heat → Electric pulp test, with a 5 minute interval between tests. 
Cold Pulp Testing (Refrigerant Spray) 
The cold test was performed by using a refrigerant spray containing propane-butan mixture 
(Endo-Frost; Roeko, Coltene Whaledent, Germany). Cold spray was applied to a size 2 cotton 
pellet, and once visible frosting was observed, it was placed on the tooth surface. The cotton 
pellet was held in contact for up to 15 seconds or until the patient responded.  
Heat Pulp Testing (Frictional Heat) 
The heat test was performed using a sterile rubber cup (NAIS, Sofia, Bulgaria) mounted on a 
low-speed contra-angle handpiece at a constant speed setting. The rubber cup was applied 
with light, consistent contact to predefined areas of the tooth surface without water cooling, 
and the stimulus was stopped when the patient responded or after a maximum of 7 seconds. 
Electric Pulp Testing 
The electric pulp test was performed using an apex locator integrated with a built-in pulp 
tester (Ai-Pex; Woodpecker, Guilin, P.R. China). A small amount of toothpaste was applied to 
the 2 mm electrode tip before placing it on the predefined area on the tooth surface. The 
device was set to the lowest current increase rate to precisely determine the numeric value 
at which the patient first perceived the stimulus. This value was recorded. The contralateral 
tooth was used as a control. If the response from the test tooth was equal to or less than the 
control tooth, the test result was recorded as 'response present.' If the response was higher 
or no response was observed up to the maximum value of 80, the result was recorded as 
'response absent.' 
Reference Standard: Pulpal Bleeding During Access Cavity Preparation 
Immediately after completion of the pulp tests, local anesthesia (Ultracain D-S Forte, Sanofi, 
Paris, France) was administered to the teeth, and the teeth were isolated with rubber dams. 
A standard endodontic access cavity was prepared by a different researcher who was blinded 
to the pulp test results. The presence or absence of pulpal bleeding in the pulp chamber was 
recorded by direct visual inspection under 3.5x magnification (dental loupes). Teeth were 
classified as vital when bleeding was observed within the pulp chamber. Teeth were classified 
as non-vital when no bleeding was present in the pulp chamber. In cases where bleeding was 
observed only within the root canal(s) without bleeding in the pulp chamber, or where 

bleeding was not present in all canals of a multi-rooted tooth, the tooth was classified as 
partially necrotic and recorded as non-vital. Following determination of pulp status, root canal 
treatment was performed. Teeth with uncertain pulp status were excluded from the study. As 
the bleeding status of the pulp were recorded as present or absent, no indeterminate test 
result category was defined or analyzed in this study. 
Data Collection and Management 
• 
Pulp test outcomes were recorded dichotomously (present/absent). Electric pulp test 
threshold value was additionally recorded. 
• 
Reference standard outcome was recorded as bleeding present/absent and final 
classification vital/non-vital. 
• 
Data were stored in a secured dataset accessible only to the research team. Identifiers 
were minimized; any exported documents may be redacted for confidentiality. 
Risks, Burden, and Safety 
Pulp tests may cause transient pain/discomfort. Local anesthesia and access cavity 
preparation were performed as part of indicated clinical care. Any adverse events or 
unexpected issues were managed according to standard clinical protocols and documented as 
appropriate. 
 
 
 
 

Statistical Analysis Plan (SAP) 
 
The statistical analyses and decision rules described below were pre-specified before study 
initiation and the SAP was finalized prior to database lock and before any outcome analyses 
were performed. Data were analyzed in IBM SPSS Statistics (Version 23). One tooth per 
participant was included. Teeth with uncertain reference standard classification were 
excluded from analysis. Index test results (cold, heat, and electrical pulp test) were coded as 
binary outcomes (response present/absent). The reference standard was pulpal bleeding 
during access cavity preparation, coded as vital (bleeding present) or non-vital (bleeding 
absent). Partial necrosis (bleeding limited to root canals without pulp chamber bleeding 
and/or not present in all canals) was classified as non-vital. 
For each index test, 2×2 contingency tables were constructed to identify true positives, false 
positives, true negatives, and false negatives. Primary performance measures were PPV and 
NPV with 95% confidence intervals (Wilson score method). Secondary measures included 
false-positive and false-negative proportions, sensitivity, specificity, and overall accuracy (all 
with 95% Wilson CIs), as well as prevalence of vitality based on the reference standard. 
Agreement between each index test and the reference standard was assessed using Cohen’s 
kappa. Differences among the three tests were assessed using Cochran’s Q test, and pairwise 
comparisons were conducted using Bonferroni-adjusted Z tests. Statistical significance was set 
at p<0.05. 
 
 
References 
1. 
Villa-Chavez CE, Patino-Marin N, Loyola-Rodriguez JP, Zavala-Alonso NV, 
Martinez-Castanon GA, Medina-Solis CE. Predictive values of thermal and electrical 
dental pulp tests: a clinical study. J Endod. 2013;39(8):965–9. 
2. 
Gopikrishna V, Tinagupta K, Kandaswamy D. Evaluation of efficacy of a new 
custom-made pulse oximeter dental probe in comparison with the electrical and 
thermal tests for assessing pulp vitality. J Endod. 2007;33(4):411–4. 
3. 
Dastmalchi N, Jafarzadeh H, Moradi S. Comparison of the efficacy of a custom-
made pulse oximeter probe with digital electric pulp tester, cold spray, and rubber cup 
for assessing pulp vitality. J Endod. 2012;38(9):1182–6. 
4. 
Petersson K, Soderstrom C, Kiani-Anaraki M, Levy G. Evaluation of the ability of 
thermal and electrical tests to register pulp vitality. Endod Dent Traumatol. 
1999;15(3):127–31. 
5. 
Weisleder R, Yamauchi S, Caplan DJ, Trope M, Teixeira FB. The validity of pulp 
testing: a clinical study. J Am Dent Assoc. 2009;140(8):1013–7. 
6. 
Hazard ML, Wicker C, Qian F, Williamson AE, Teixeira FB. Accuracy of cold 
sensibility testing on teeth with full-coverage restorations: a clinical study. Int Endod J. 
2021;54(7):1008–15. 

7. 
Salgar AR, Singh SH, Podar RS, Kulkarni GP, Babel SN. Determining predictability 
and accuracy of thermal and electrical dental pulp tests: An in vivo study. J Conserv 
Dent. 2017;20(1):46–9. 
8. 
Georgopoulou M, Kerani M. [The reliability of electrical and thermal pulp tests. A 
clinical study]. Stomatologia (Athenai). 1989;46(5):317–26. 
9. 
Evans D, Reid J, Strang R, Stirrups D. A comparison of laser Doppler flowmetry 
with other methods of assessing the vitality of traumatised anterior teeth. Endod Dent 
Traumatol. 1999;15(6):284–90. 
10. 
Kamburoğlu K, Paksoy CS. The usefulness of standard endodontic diagnostic 
tests in establishing pulpal status. The Pain Clinic. 2005;17(2):157–65. 
11. 
Mejare IA, Bergenholtz G, Petersson K, Tranaeus S. Estimates of sensitivity and 
specificity of electric pulp testing depend on pulp disease spectrum: a modelling study. 
Int Endod J. 2015;48(1):74–8. 
 
 
 

Participant Information Sheet and Informed Consent Form 
INFORMED CONSENT FORM 
Version date: 05.06.2024   Version no: 02 
 
The title of this study is “Diagnostic Accuracy of Cold, Heat, and Electric Pulp Tests: An In Vivo 
Study”. This is a specialty (residency) thesis research project. The aim of the study is to 
determine the predictive values and accuracy of thermal (heat/cold) and electric pulp tests (a 
device that tests tooth vitality by delivering specific signals to the tooth) in assessing the 
vitality of the dental pulp, which is the living tissue inside the tooth. 
Root canal treatment is performed when there is deep decay in a tooth or when the tooth is 
infected. In this research method, tests used to determine the vitality status of teeth that have 
already been indicated for root canal treatment will be performed. These tests are thermal 
(cold and heat) and electrical tests. The tests specified in this study will be applied to your 
tooth/teeth, and afterwards root canal treatment will be performed as part of your routine 
care (outside the research procedures). All of the tests specified are tests that are successfully 
used in routine clinical practice. 
In this study, pulp sensibility tests will be applied in sequence to the tooth for which root canal 
treatment has already been indicated. All pulp sensibility tests (cold, heat, and EPT) will be 
performed first on an asymptomatic, functional contralateral tooth without previous 
endodontic treatment (or, if unavailable, on a suitable adjacent tooth) to establish a baseline 
response before testing the test tooth. First, a cold spray test (application of a cold agent 
sprayed onto sterile cotton), then a frictional heat test (stimulation of the living tissue by 
generating heat on the tooth with a rotating rubber cup), and finally an electric pulp test will 
be applied. A 5‑minute interval will be allowed between tests so that the results do not affect 
each other. Your tooth will first be isolated from oral fluids (with cotton rolls and an air spray). 
The cold spray will be sprayed onto a small piece of cotton and placed on the front middle 
surface of your tooth and applied for a maximum of 15 seconds. Then, a polishing rubber cup 
(used for polishing dental fillings) will be rotated on your tooth for a maximum of 7 seconds. 
Finally, after applying a thin layer of toothpaste to the tooth as a conducting medium, the 
electric pulp test probe will first be placed on the front middle surface of the tooth opposite 
the tooth to be tested, and electrical current will be started at the lowest level. Based on the 
numerical value displayed by the device and your perceived pain/discomfort, the numerical 
value will be recorded; then, according to this value, the electric pulp test will be applied to 
the tooth to be tested in the same way. In all three tests, the test will be stopped and the 
results will be recorded as soon as you feel any pain/discomfort/tingling. If you do not feel any 
pain/discomfort/tingling within the specified time, the test will still be stopped and your test 
result will be recorded as ‘response absent’. After the tests are completed, an access cavity 
(opening) used in routine treatment will be prepared and your test result will be compared 

with the bleeding status of your tooth. After preparation of the access cavity and recording of 
vitality status, standard root canal treatment will be performed for your tooth/teeth. 
Possible undesired effects and risks are as follows: The tests to be applied do not cause harm 
to the teeth or surrounding tissues. 
At the end of the study, the responses of three different tooth sensibility tests that are 
routinely used in dentistry will be evaluated in different clinical conditions, and it will be 
compared which test yields results closer to the diagnosis. Thus, it will be determined which 
of these routinely used tests is more helpful for diagnosis. This study may provide guidance 
regarding the comparison and accuracy of tests that clinicians can use to establish a correct 
diagnosis in the clinic. 
The study duration is 6 months. The expected participation time for volunteers is 
approximately 1 hour (time to complete the tests and the subsequent root canal treatment). 
The institution where the study will be conducted is the Department of Endodontics, Istanbul 
University Faculty of Dentistry. A total of 175 volunteers will be included in the study. 
There are alternative treatments outside the research method; namely, root canal treatment 
can be performed on the tooth/teeth already indicated for root canal treatment without 
applying the above tests. 
Your participation in the research is voluntary. You may refuse to participate or withdraw from 
the study at any time without any penalty or sanction and without losing any of your rights. If 
you do not accept participation or if you are removed from the study program for any reason, 
there will be no disruption in your treatment. Records that could reveal your identity will be 
kept confidential, will not be disclosed to the public, and the study results will not be published 
in a way that could lead to identification. If such a situation arises, your additional consent will 
be obtained. Even if the study results are published, your identity will remain confidential. 
Monitors, auditors, the ethics committee, the institution, and other relevant health 
authorities may have direct access to the volunteer’s original medical records; however, this 
information will be kept confidential. If new information related to the research that may 
affect your willingness to continue participation is obtained, you or your legal representative 
will be informed. You will not incur any financial responsibility for study-related expenses, and 
no payment will be made to you. 
Dear Dr. Ecem Erden, I have been informed that a medical research study will be conducted 
at the Department of Endodontics, Istanbul University Faculty of Dentistry, and the above 
information regarding this research has been provided to me. After receiving this information, 
I have been invited to participate in such a study as a “volunteer”. If I participate in this study, 
I believe that great care and respect will be shown for the confidentiality of my personal 
information that should remain between my physician and me. I have been given sufficient 
assurance that my personal information will be protected with due care during the use of 
study results for educational and scientific purposes. 

During the conduct of the project, I may withdraw from the study by informing the researchers 
without stating any reason. In addition, I may be excluded from the study by the researcher 
provided that no harm is caused to my medical condition. 
I will not assume any financial responsibility for the study-related expenses. No payment will 
be made to me. 
The necessary assurance has been provided that, should any health problem occur for any 
reason directly or indirectly arising from the research procedures, all required medical 
interventions will be provided (and I will not bear any financial burden related to these 
medical interventions). 
If I experience any health problem during the study, I know that I can contact Dr. Ecem Erden 
at any day and time at the following address and phone number: Istanbul University Faculty 
of Dentistry, Süleymaniye, Prof. Dr. Cavit Orhan Tütengil St. No:4 D:6, 34116 Fatih/Istanbul, 
Türkiye. Phone: +90 5***. 
I am not obliged to participate in this research and I may choose not to participate. I have not 
encountered any coercive behavior regarding my participation. If I refuse to participate, I know 
that this will not harm my medical care or my relationship with my physician. 
After having sufficient time to consider, I have decided on my own to take part in the above-
mentioned research project as a “volunteer”. I accept this invitation of my own free will, 
having been adequately informed about the outcomes and effects of the research, without 
any pressure, and without any promise of benefit. 
A copy of this signed form will be given to me. 
I have read all explanations in this informed consent form. Written and verbal explanations 
about the research described above were provided to me by the physician named below. I 
know that I am participating voluntarily, without any pressure or coercion, and that I may 
withdraw from the study at any time with or without providing a reason. 
Volunteer’s Full Name / Signature / Date 
 
Name / Signature / Date of a competent researcher in the study team 
 
Name / Signature / Date / Address / Phone of the witness to the consent process (if 
required) 
 
Legal representative (mother/father) – Full Name / Signature / Date / Address / Phone (if 
required)