Establishing a biobank for road traffic accident victims in Iran as a cohort study of stress related biomarkers

Establishing a biobank for road traffic accident victims in Iran as a cohort study of stress related biomarkers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Establishing a biobank for road traffic accident victims in Iran as a cohort study of stress related biomarkers Leila Vahedi, Mostafa Farahbakhsh, Aysan Mohammad-Namdar, Homayoun Sadeghi-Bazargani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8409982/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chronic stress plays a contributing role in occurrence of traffic crashes and development of post-traumatic stress disorder (PTSD). Due to lack of a biobank for traffic injury victims in Iran, this study was conducted to develop a protocol for the design and establishment of a biobank for these victims. Methods This prospective cohort study will be conducted for a period of two years on individuals hospitalized due to traffic injuries at Imam Reza hospital in Tabriz University of medical Sciences/Iran. After extracting the details of individuals recorded in the Iranian Integrated Road Traffic Injury Registry SYSTEM (IIRTIRS), questionnaires on demographic characteristics, hair status, and life stressor events, and biological samples including hair and blood will be collected. Participants will be followed up after two month to complete the follow-up questionnaires. Qualitative and quantitative data (like hair cortisol levels) will be analyzed using the Chi-square test and ANOVA, respectively. Pearson correlation will be used to assess the association between hair cortisol levels measured by the enzyme-linked immunosorbent assay (ELISA) and chromatography methods. Conclusion The biobank of traffic crash victims in Northwest of Iran was designed and will be initiated by the Traffic and Health Hub, with collaboration from university-affiliated research centers and hospitals. As the first biobank of its kind in the country, its protocol has the potential to function as a blueprint for establishing similar biobanks in other developing countries. Biobank Hair Cortisol Stress Road accident Figures Figure 1 Figure 2 Figure 3 Introduction In recent years, the increasing mortality and injuries due to road traffic accidents have remained one of the leading causes of disability and a major public health concern. (1) Annually, road traffic accidents result in injuries to 20 to 50 million individuals worldwide, with approximately 1.19 million fatalities. (2) The fatality rate due to traffic injuries has decreased in developed countries, whereas it continues to increase in low- and middle-income countries, such that nearly 90% of global deaths from traffic injuries occur in these regions. (3) Iran is among the countries with the highest rates of traffic accident fatality and disability worldwide. (4) In addition to causing death and disability, traffic accidents can also lead to a variety of social, economic, and psychological consequences. (5) Post-traumatic stress disorder (PTSD) is considered one of the most common mental health consequences of traffic accidents. (6) Epidemiological studies indicate that not all individuals exposed to the same traumatic event will develop PTSD; rather, individual vulnerability plays a significant role in this regard. This vulnerability can be either genetic or acquired after birth. (7) Therefore, identifying the factors that contribute to traffic accidents as well as PTSD is of great importance. Among these factors, environmental influences, demographic characteristics, and individual factors such as stress can be highlighted. (8,9) Stress is one of the risk factors for chronic diseases that can lead to both physical and psychological issues, and manifests in two forms: acute and chronic. Acute stress, through its impact on the hypothalamus and the transmission of nerve impulses to the central part of the adrenal gland, results in the secretion of catecholamine hormones, that is, epinephrine and norepinephrine. (10) In contrast, in chronic stress, the hypothalamic-pituitary-adrenal (HPA) axis is activated, ultimately yielding the secretion of cortisol hormone from the adrenal cortex. This hormone has long-term effects and is involved in the pathophysiology of a wide range of physical and psychological problems. Cortisol has recently become a key focus in psychoneuroendocrinology research. (11) Although it can be detected in blood, urine, and saliva, its levels fluctuate significantly over a 24-hour period (12) . In contrast, hair provides a reliable medium for assessing cortisol levels (11) , as cortisol enters hair primarily through passive diffusion. Given that hair grows at a relatively constant rate of 1 centimeter per month, each centimeter reflects roughly one month of cortisol production, thereby mitigating the issue of cortisol fluctuations (13) . Moreover, by segmenting hair samples into different sections and analyzing them separately, a retrospective timeline of exposure to substances and the onset of disease can be determined (14) . Accurate identification of the factors influencing traffic injuries and PTSD requires the standard collection of biological samples and the analysis of a large volume of biological data. Biological samples are valuable and non-renewable resources, particularly when accompanied by follow-up and clinical care (15) . Each biological sample should be considered not only as a small genetic and molecular resource for research programs but also as a database for advanced patient treatments based on personalized medicine. Personalized medicine, based on the genetic characteristics of each individual, focuses on treating people (16) . One of the key tools for research on personalized medical is the study of human biological samples, including hair, nails, DNA, and serum. The establishment of biobanks facilitates access to biological samples (17) . Biobanks consist of tissues, organs, plasma, saliva, DNA, RNA, proteins, living cells, genetic material from living organisms, hereditary information, and functional data related to biological systems, all preserved for research purposes over an extended period. Thus, biobanks act as the cornerstone of personalized medicine (18) . Their benefits are realized when they are established with careful attention to project objectives, sample quality control, high-quality data generation, data integrity, security, confidentiality, and the management and sharing of samples—all of which are critically important (17) . Considering the role of chronic stress in occurrence of traffic accidents and their severe consequences, both of which impose significant functional and financial burdens, research on biological samples is crucial to identify individuals at risk for such accidents and to prevent post-crash complications. Therefore, given the lack of previous efforts to collect biological samples from traffic accident victims, the establishment of a biobank for these individuals in Iran is indispensable. This study aims to design and create a protocol for a biobank of road traffic victims. Methods and analysis This prospective cohort study will be conducted over two years on individuals hospitalized following traffic accidents at Imam Reza hospital (as educational and referral hospital) in Tabriz University of medical Sciences/Iran. The study is aligned with the Tabriz University of Medical Sciences' Road Traffic Injury Registry Program and the Persian Traffic Health and Safety Cohort Program, contributing to the development of both initiatives. Figure 1 illustrates the overall study algorithm. The methodology of the present study is structured in two sections: the establishment and implementation of the biobank. Establishment of the biobank program for the traffic accident victims The first step in establishing and implementing the biobank is its conceptualization, aimed at identifying and defining the foundational structures of the program. For the conceptualization of the traffic accident victim biobank, an organizational structure for the program was first established. Accordingly, the program is implemented under the supervision of the Vice Chancellor for Research and Technology at Tabriz University of Medical Sciences, within the RTIRC. The program comprises committees such as the Executive Committee, Scientific Committee, Software and Quality Control Committee, and the Safety and Sample/Information Access Committee. The roles of the program’s key members are detailed in Table 1. Table 1. Responsibilities of Biobank Key Members Subsequently, the management of the biobank's operational activities, the process for data sharing, and the policies for utilizing the biobank's resources for traffic accident victims were defined. The ten key policies are outlined in Table 2. Data sharing will occur through the website of the RTIRC, where an announcement will be issued regarding the use of samples and data, in accordance with the conditions and regulations specified in the ethical guidelines. The ownership of these samples is held by the RTIRC at Tabriz University of Medical Sciences. Table 2. Policies of Biobank Program Finally, the necessary physical infrastructure for implementing the program was determined and prepared. In this regard, Imam Reza hospital was designated for the first phase that is sample collection from individuals admitted to this hospital following a traffic accident, the HTSC for the follow-up phase that is re-sampling, psychiatric consultation, and other necessary assessments, and the central biobank of Tabriz University of Medical Sciences for storing blood samples. Implementation of the biobank program for the traffic accident victims The program will be executed in two phases. In the first phase, individuals hospitalized due to traffic accidents at Imam Reza Educational and Medical Center in Tabriz will be identified by biobank experts stationed at the center. After assessing inclusion and exclusion criteria, their names and details will be recorded in the Traffic Accident Registration System. Subsequently, with ethical considerations in place, participants will complete demographic questionnaires, hair condition forms, and questionnaires on stress-inducing life events. Finally, biological samples (hair and blood) will be collected in accordance with prior instructions and training. To follow up and screen the traffic accident victims, biobank experts at the RTIRC will make phone calls two months after the road traffic. During these calls, follow-up forms will be completed. If a participant scores PTSD ≥ 3 or PHQ ≥ 5, they will be invited to the HTSC for further evaluation. At the clinic, after completing the relevant questionnaires, participants will undergo a driving test using a simulator, an assessment via the Vienna Test System, and a consultation with a psychiatrist. If victims meet the criteria for PTSD as outlined in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition), they will be classified as PTSD-positive. Finally, biological samples (hair and blood) will be collected again from participants, both PTSD-positive and PTSD-negative. Participants and sample size The participants in this study include individuals hospitalized at Imam Reza Hospital due to traffic accidents, whose information is recorded in the Iranian Integrated Road Traffic Injury Registry (IIRTIR) within Region two of the National Healthcare Map. Individuals eligible for inclusion in the study are those aged over 18 years who express willingness to participate in the research. However, several exclusion criteria apply: individuals unwilling to cooperate, those with hair shorter than 3 centimeters, those who have used systemic or topical steroids on the scalp within the past three months, and individuals who are currently pregnant or have been pregnant within the past three months. Additionally, individuals with severe illnesses such as cancer, HIV, or autoimmune disorders, those who have abused alcohol or substances within the past three months, and individuals with current major depressive disorder (MDD) accompanied by psychotic symptoms and suicidal thoughts are excluded. Participants with bipolar disorder or suicidal thoughts within the past three months are also excluded. The sample size is based on a study by Russell (15) , with 20 participants estimated per month. Given the planned two-year study duration, a total of 600 participants (400 cases in the initial phase and 200 cases in the follow-up phase) are expected. Participants will be selected using a non-random convenience sampling method. Data and biological sample collection In this study, data such as demographic characteristics, hair-related information, and details of life stressor events will be collected through questionnaires. Hair and blood samples will be collected by biobank experts at both Imam Reza hospital and the RTIRC. Moreover, psychiatric examinations will be conducted at the RTIRC by a psychiatrist through interviews with the patients. The details of each data collection method are described below. Data collection via questionnaires After obtaining informed consent and clearly explaining the study objectives to the participants, and considering the inclusion and exclusion criteria, the questionnaires will be filled out at Imam Reza hospital during the first phase, and at the RTIRC, during the two-month follow-up screening phase. The questionnaires will be stored at the RTIRC. Questionnaire on demographic information and hair status of victims To prepare the questionnaire, methods such as literature review, examining biobank registration centers in other countries, conducting a pilot study, and brainstorming were utilized. Eventually, a semi-structured questionnaire was designed. This questionnaire includes a series of questions in three areas: demographic information, hair status, and previous accidents of the victims. The questions cover aspects such as age, gender, education, marital status, weight, height, alcohol consumption, cigarette smoking, contraceptive use, menopausal status, pregnancy status, breastfeeding status, underlying medical conditions, regular use of medications, hair dyeing, other hair treatments such as perming, hair extensions, or straightening, frequency of hair washing within a week, hours of direct sun exposure to the hair in a day, number of severe accidents experienced, hair cortisol levels, and timing of sample collection. The validity of this questionnaire was confirmed through content validity and face validity methods. Questionnaire on stress-inducing life events of victims This questionnaire pertains to the ranking of stress-inducinglife events and measures the amount of stress an individual has experienced over the course of a year. The Holmes and Rahe Stress Scale includes 43 life events, with each assigned a numerical rating. Each event is considered a unit of life change with a different weight in generating stress. If an event has occurred more than once for the individual, the corresponding score is doubled before summing the total to derive the final score. For a score between 11 and 150, there is a very low to moderate likelihood that the individual will become ill due to stress in the near future. For a score between 150 and 299, the likelihood of illness due to stress increases to moderate or high. For scores between 300 and 600, there is a high to very high probability that the individual will become ill due to stress. Maleki et al. conducted a cross-sectional descriptive study on the population of Tabriz. Considering cultural and social differences, they modified the Holmes-Rahe Stress Questionnaire and developed a 50-item questionnaire assessing stress-inducing events (19) . The evaluation of participants will be based on this adapted questionnaire, focusing on stressful events experienced over the past year. The database scheme This study uses a relational database developed in Microsoft Access to manage and organize data collected from participants in a prospective cohort study on chronic stress and its role in accidents and PTSD. The database scheme is designed to facilitate the integration of multiple data types, including demographic information and Stressful life events. The database consists of four primary tables linked by a unique identifier, the National ID Number. This identifier is used to ensure that data from various sources can be accurately combined for each individual. The four tables in the database are: 1. Demographic information, hair status, and medication consumption: This table stores participant demographics (e.g., age, gender, occupation, and etc.) and details on hair status (e.g., hair washing frequency, hair dying) and medication consumption. 2. Stressful life events experienced by the individuals during the last three spans: This table records stressful life events and the scores assigned to each question participants experienced during the last three spans i.e. three months, six months and 12 months including events like job loss, family death, financial problems, and etc. Due to the field limitation in Microsoft Access (i.e. less than 255 fields per table), the second and third tables, related to stressful life events, contain the remaining questions to accommodate all questions appropriately. Consequently, the data from stressful life events is spread across several tables, guaranteeing that each participant’s data is registered in full. Each table includes a unique AutoNumber field named ID, which serves as the primary key for data entry and ensures accurate linking between tables. Data types of the fields in these tables are "Short Text," "Yes/No," and "Number" corresponding to responses on the questionnaires. Data sources include structured questionnaires (on demographics and stressful life events), biological samples (hair and blood for measuring cortisol levels), and hospital records from Imam Reza Hospital, Tabriz registered in the IIRTIRS. Data entry is carried out by trained research staff, following strict protocols to assure accuracy and consistency. The database supports data integration from the data sources, providing a comprehensive view of participant experiences and stress biomarkers. Quality control includes automated data validation checks, regular audits, and the use of data-entry protocols to minimize human error. Data is securely stored, and access is restricted to authorized personnel to maintain confidentiality and integrity. The PTSD diagnostic questionnaire This questionnaire is a follow-up form for traffic accident survivors aged 18 and above, focusing on events within the past month. It is to be completed via phone and includes five yes/no questions, with the total score determined by summing responses. Higher scores indicate greater severity of the disorder. Individuals scoring three or higher are referred to the THSC for further evaluation. The PHQ-9 questionnaire This questionnaire is a widely used tool over the past two decades for assessing depression. It consists of ten questions, with the first nine corresponding to DSM-5 criteria for major depressive disorder, such as sadness and hopelessness, loss of interest or pleasure, sleep disorders, fatigue or low energy, appetite changes, feelings of worthlessness or guilt, agitation, difficulty in concentrating, and suicidal thoughts In the last question, the overall functioning of the individual is assessed. Each question has four response options: "not at all," "several days," "more than half the days," and "nearly every day," scored from 0 to 3, respectively. To diagnose depression, at least five questions must be marked as "more than half the days" or "nearly every day." To calculate the total score of the PHQ-9 questionnaire, the scores for each question are summed up, and a new variable is generated. If the total score exceeds four, it signifies depression. The reliability of this questionnaire with a Cronbach alpha of 88% and content validity of 74% has been confirmed in the study by Dadfar et al. (20) Data collection through behavior measurement devices The Vienna Test System (VTS) VTS is a computerized tool designed for psychological, neurological, and transportation evaluations, offering high objectivity and precision, including factors that traditional pen-and-paper tests cannot measure. The indigenous driving performance assessment device, developed by the Traffic Research Center at Tabriz University of Medical Sciences, is based on data from Iranian participants. Before the main tests, individuals perform practice exercises to familiarize themselves with the system. Tests in this study include assessments for peripheral vision, reaction time, and other relevant measures. Driving test in a simulator The Sahand Driving Simulator was developed in 2018 by the Traffic Research Center at Tabriz University of Medical Sciences. This simulator consists of a real cabin based on a Peugeot 405, one of the most commonly used cars in Iran, and three 43-inch monitors. The software used in the simulator is openDS, an open-source program that has been enhanced to meet the research needs of the RTIRC. The scenario used in this study includes driving in three different settings: rural, urban, and highway environments. The total driving time for the scenario ranges from 23 to 30 minutes. Notably, all drivers first complete a five-minute practice scenario to make themselves familiar with the simulator before starting the main scenario. The simulator assesses various driving functions designed to evaluate driving abilities related to response to unexpected events, collision avoidance behavior, total brake reaction time, simulator performance index, driving performance measures, and continuous time to collision. These skills are evaluated through the scenarios, providing an assessment of each individual’s driving performance. The validity and reliability of the driving simulator have been confirmed by the RTIRC. Collection of biological samples Once informed consent is obtained and the objectives are clearly explained to the participants, hair and blood samples will be collected. The sample collection will be performed by trained experts, taking into account the inclusion and exclusion criteria as well as ethical considerations. Procedure of hair sample collection Wearing gloves and using sterilized scissors, biobank experts will collect approximately 100 hair strands from the posterior region of the scalp, with each hair strand being about the thickness of a pencil and 3 cm in length. After identifying the proximal end of the hair, it will be placed inside a 5*5 cm piece of thick aluminum foil (Figure 2). The foil will then be placed inside a 5*7 cm ziplock bag along with 10 to 15 desiccants or silica gel packets. The ziplock bags will be stored in Frozen (-20°C). Each ziplock bag will be labeled with a unique code assigned to each patient, which must match the codes both on the questionnaires and in the IIRTIRS. Finally, the hair samples must be stored in a dry and dark environment, away from direct sunlight, at the HTSC of the RTIRC. Hair samples should not be stored in a refrigerator or freezer. It is worth mentioning that scalp hair is the preferred sample; however, if scalp hair is not available, particularly in men, hair samples may be collected from other parts of the body, such as the arms, chest, underarms, beard, and legs. The specific area from which the hair was collected must be recorded in the corresponding section of the hair archiving list under "Notes." The collection of hair samples requires careful attention to the donor's privacy. Figure 2. Procedure of hair Sampling Procedure of blood sampling Blood samples will be collected by a trained individual wearing gloves. A total of 10 cc of venous blood will be drawn into ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes. The tubes will be gently inverted to prevent the formation of blood clots. After collecting the blood sample in EDTA tubes, it is centrifuged, then the white blood cells (buffy coat layer) are extracted. Immediately after separation, they are aliquoted and placed in special cryovial tubes. Protective solutions such as TE buffer are added to help stabilize the DNA, and then the samples are frozen at -80°C, where DNA will be extracted from the blood as soon as possible (based on approved projects) (Figure 3). The collection of venous blood samples is contingent on sufficient funding, as well as the collaboration of Imam Reza hospital and the central biobank. Each EDTA anticoagulant tube will be labeled with a unique code assigned to each patient. These codes must also match the codes both on the questionnaires and in the IIRTIRS. Figure 3. Storage area for blood samples in the central biobank Psychiatric examinations Psychiatric examinations will be conducted during the follow-up phase by a psychiatrist for participants who have visited the HTSC. Following the examinations, the participants will be classified into three groups: the first group consists of participants whose post-traumatic stress disorder (PTSD) is positive in both the screening and the psychiatrist's examination. The second group includes participants whose PTSD is positive in the screening but negative in the psychiatrist's examination. The third group comprises participants whose PTSD is negative in both the screening and the psychiatrist's examination. Subsequently, the results of the questionnaires and hair cortisol levels will be measured and compared among the three groups. The measurement of hair cortisol will be done using the ELISA method and Liquid chromatography–mass spectrometry (LC–MS). Quality control Several approaches have been considered by the central study team to ensure and maintain the quality of the follow-up implementation process, which include: 1. Evaluation of the quality of follow-up implementation on the Central Biobank Quality Assurance and Control Committee’s behalf at six-month intervals. 2. Monthly review of the accuracy of completed follow-up forms and data cleaning by examining forms that have been recorded as incomplete, incorrect, or duplicate. 3. Monthly verification of the accuracy of form completion for each field by conducting follow-up phone calls with at least 10% of participants in each field, selected randomly, by the Biobank Quality Control Committee. 4. Completion of self-assessment forms by interviewers and other follow-up team members at one-month intervals. 5. Monthly evaluation of the required equipment and human resources. 6. Assessment of the quality of blood sample preparation and storage, and other biological samples. 7. Review of the quality and accuracy of the work performed by the biobank experts. Maintenance of ultra-low temperature freezers (-80 degrees Celsius ) in the biobank Ultra-low temperature (ULT) freezers store contents at −80°C and are among the most critical equipment in a biobank laboratory. The primary principles for extending the lifespan and ensuring the cooling system's efficiency, as well as preserving sample quality, include installing digital temperature monitoring systems and recording daily temperature fluctuations using dedicated software with notification capabilities. Temperature fluctuations inside the freezer exceeding ±1.5°C are undesirable. The freezer should be cleaned every six months and undergo an annual freeze and defrost cycle, during which the freezer is emptied, powered off, cleaned, and restarted. In addition, the frost on the freezer door should be removed weekly, and the door gasket should be inspected regularly. Reporting All team members must promptly deliver critical and significant reports to the biobank manager (via phone). The Quality Control Committee is required to submit a monthly report of the quality control assessment results to the biobank manager. Interviewers and other members of the implementation team must report any challenges and limitations in executing the guidelines to the biobank manager. Revision, Rescheduling, and Retraining Based on the quality control results, the need for retraining follow-up team members should be considered. Retraining will be conducted by the biobank manager. It is also important to note that the number of cohort participants decreases over time due to deaths or withdrawal from the study, highlighting the necessity for the biobank manager to adjust the schedule accordingly. The biobank manager is responsible for reviewing the necessary equipment and human resources, identifying any shortages or issues, and reporting them to the central team at monthly intervals or, if needed, immediately for appropriate action. If the implementation of this protocol becomes unfeasible or fails to meet the study's objectives, the follow-up activities should be halted, and a new protocol should be developed. Biobank’s database management system Finally, the information obtained from the victims will be entered into the IIRTIRS (International Institute for Road Traffic Incident Reporting System). Once the questionnaires are completed, the data will be entered into the database management system, which links questionnaire responses with biological sample data using a unique identifier (e.g., national code) assigned to each victim. This guarantees the proper tracking and management of each participant’s data across all records. The registry experts at the RTIRC will conduct the data quality control. Additionally, one data committee member will act as a liaison between the database management system and the user. Measurement of cortisol concentration in hair To measure cortisol concentration in hair, two methods will be utilized: ELISA and chromatography, based on the study by Russell et al. (13) . The summarized steps for the ELISA method, according to Kirschbaum et al., are as follows: Hair washing, hair powdering, solvent extraction, incubation, centrifugation during extraction, evaporation, reconstitution with solvent, vortexing, use of ELISA and consideration of cross-reactivity, centrifugation during ELISA and consideration of wavelength when measuring cortisol. In the ELISA method, ALPCO's of ELISA kits (New Hampshire, USA) will be used. Liquid chromatography-mass spectrometry (LC-MS/MS), or the chromatography method, will be performed at Faculty of Pharmacy/Tabriz University of Medical Sciences/Iran. DNA extraction and genetic analysis of blood samples will also be carried out based on similar projects. Statistical analysis This study is designed to collect data on road accidentvictims and serves as an infrastructure research project. The information gathered in this study will be utilized in future research projects based on their specific objectives.In general, the study will assess demographic data, trauma-related questionnaires, stress levels, the presence of PTSD, and hair cortisol levels across three groups. To compare qualitative data, such as gender and the presence or absence of PTSD among the three groups, the Chi-square test will be used. For comparing quantitative data, such as stress questionnaire scores and hair cortisol levels among the three groups, analysis of variance (ANOVA) will be used.The Pearson correlation test will be applied to assess the correlation of hair cortisol levels between the ELISA and chromatography methods. Logistic regression analysis will then be used to predict the odds of developing PTSD. P-value< 0.05 will be considered statistically significant. Discussion Road traffic accidents and their consequences are among the leading causes of mortality and disability across various age and gender groups in low- and middle-income countries (3) . Genetic factors are a key determinant of individuals' vulnerability to traffic accidents and the complications following them (7) . Hence, research aimed at uncovering the genetic mechanisms contributing to traffic accidents and their consequences is of critical importance. Biobanks serve as a reliable infrastructure for collecting and preserving biological samples. The findings derived from these biobanks can provide valuable evidence for understanding the genetic factors influencing the risks and outcomes of traffic accidents, enabling researchers worldwide to make valid and evidence-based decisions (17,21) . This study provides detailed information on the design and establishment of the biobank for road accidentvictims, a pioneering initiative in the country. The biobank will be launched and executed by the RTIRC, which has been officially designated as the national reference hub for traffic and health by the relevant authorities. The purpose of this biobank is to collect, store, and analyze biological samples, such as hair and blood, from individuals hospitalized due to traffic accidents. The RTIRC, recognized as the site of the country’s first traffic cohort (22, 23) and a WHO Collaborating Center, is a leading institution in traffic safety in Iran. Therefore, during the implementation of the biobank, it can leverage its previous experiences and international collaborations to promote the program. As part of this project, protocols and guidelines for establishing the biobank will first be prepared, and data collection will be carried out accordingly. Subsequently, universities nationwide will be notified about the existing data to encourage their participation in future biobank-related projects across various disciplines. International researchers will also be invited to collaborate, leveraging their expertise for the development and implementation of upcoming biobank projects. To maintain and enhance the scientific and operational quality of the biobank, continuous updates to protocols, ongoing staff training, expansion of sampling centers, collaboration with international biobank development centers, partnerships with other national universities to establish related biobanks, and securing sustainable financial resources may be required over time. Furthermore, a comprehensive network of experts in medical genetics, traffic injuries, psychiatry, chemistry, pharmacy, epidemiology, and biostatistics is essential. Adherence to ethical and legal guidelines, strong software infrastructure, advanced hardware equipment, and adequate human resources are also critical for the optimal operation of the biobank. Therefore, the biobank program must receive full support from national authorities, as well as governmental and non-governmental organizations in these aspects. Conclusion Due to the impact of chronic stress on the occurrence of road accidents, and the severe complications that can follow such accidents, including PTSD, which imposes a significant functional and financial burden, the current research aimed at identifying individuals at risk of road accidents (including those experiencing chronic stress) and preventing the complications that may arise after road accidents (such as PTSD) appears to be important. Future studies will investigate hair cortisol as a means to reduce the incidence of road accidents and PTSD. Moreover, efforts will be made in this research to compare different methods of measuring hair cortisol, with the goal of identifying a method that is more affordable, more accessible, and highly sensitive. Strengths and limitations of this study : The study's strengths are as follows: The study was conducted on a cohort of road accident victims who were admitted to the hospital. Demographic information, hair characteristics, and stressful life events were recorded for the participants. Blood and hair samples were also prepared for analysis. The study's limitations are as follows: The majority of the women's hair samples were dyed with the second color. Due to a lack of adequate facilities, it was not possible to obtain RNA samples. Abbreviations Traffic Injury Management and Prevention Research Center Road traffic injury research center (RTIRC) Health and Traffic Safety Clinic (HTSC) Traffic and Health Reference Hub Iranian Integrated Road Traffic Injury Registry (IIRTIR) Post-traumatic stress disorder (PTSD) Hypothalamic-pituitary-adrenal (HPA) Declarations Ethics approval : The present study has been approved by the Regional Ethics Committee of Tabriz University of Medical Sciences under ethics code IR.TBZMED.REC.1401.950. Unlike other research projects, the specific type of tests to be conducted on biological samples and their potential results are not predetermined in a biobank. Therefore, ethical considerations the Declaration of Helsinki and its later are of utmost importance in biobanks. The informed consent form for the biobank of the RTIRC has been developed based on the American Society of Human Genetics guidelines regarding the storage of biological samples and data. Informed consent was obtained from all the individual participants for participation in this study. After receiving necessary explanations and addressing any potential ambiguities, individuals voluntarily and informedly sign the consent form. These signed informed consent forms are securely stored in the biobank unit of the RTIRC at the Tabriz University of Medical Sciences. The consent form explicitly states that the study is just conducted for research purposes and does not provide any diagnostic commitments. However, if any findings obtained during the research process are deemed significant for the diagnosis, disease progression, or treatment of participants, the individuals will be informed. Participation in the study is entirely voluntary, and participants may withdraw at any time. The questionnaire data and biological samples of patients will be kept strictly confidential. No names will be used on questionnaires or biological samples. The data entered into the computer system will also remain confidential, and the computer will be equipped with a security system to prevent unauthorized access to patient information. Data will be anonymized by use of codes and kept confidential. Confidentiality will be maintained even for other researchers who may use the samples and data in future studies. All data will be handled in accordance with Iranian data protection laws, guaranteeing their confidentiality. The use of biobank samples by other researchers for future studies will be conducted in accordance with the ethical charter established by the center. These researchers are required to comply with the principles of data use and confidentiality signing an agreement. Consent to participate : Participation in the study is entirely voluntary, and participants may withdraw at any time. The questionnaire data and biological samples of patients will be kept strictly confidential. No names will be used on questionnaires or biological samples. The data entered into the computer system will also remain confidential, and the computer will be equipped with a security system to prevent unauthorized access to patient information. Data will be anonymized by use of codes and kept confidential. Confidentiality will be maintained even for other researchers who may use the samples and data in future studies. All data will be handled in accordance with Iranian data protection laws, guaranteeing their confidentiality. The use of biobank samples by other researchers for future studies will be conducted in accordance with the ethical charter established by the center. These researchers are required to comply with the principles of data use and confidentiality signing an agreement. Consent to publish: Not applicable as no information or images of the participants are present in the manuscript. Data Availability: Data are available from the authors upon reasonable request. Funding: Road Traffic Injury Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. (Grant Number: 70347). Competing interests: The authors declare that they have no competing interests. Author Contributions : VL: Contributions to the conception and design of the work, control and interpretation of data, writing the manuscript FM : Examination of the survivors, interpretation of data, revision of the manuscript NA : Design and creation of the database used in the study, control data, writing the manuscript SH : Contributions to the conception and design of the work, interpretation of data, revision of the manuscript. All authors read and approved the final manuscript. Acknowledgements: The authors would like to thank all the Road Traffic Injury of Registry Team Members for collaboration in this study. References Organization WH. Global status report on road safety 2018: World Health Organization; 2019. Organization WH. Global status report on road safety 2023: summary: World Health Organization; 2023. Perel P, Ker K, Ivers R, Blackhall K. Road safety in low-and middle-income countries: a neglected research area. Inj Prev. 2007;13(4):227-. Mehryar HR, Alizadeh N, Rostampour F, Paryab S, Bab S, Garkaz O. Causes of road accidents in northwestern iran in the period 2010–2018. Int. Arch. Health Sci. 2022;9(1):14-9. Boelen PA, Eisma MC, de Keijser J, Lenferink LI. Traumatic stress, depression, and non-bereavement grief following non-fatal traffic accidents: Symptom patterns and correlates. PLoS One. 2022;17(2):e0264497. van den Heuvel LL, Stalder T, du Plessis S, Suliman S, Kirschbaum C, Seedat S. Hair cortisol levels in posttraumatic stress disorder and metabolic syndrome. Stress. 2020;23(5):577-89. Obuobi-Donkor G, Oluwasina F, Nkire N, Agyapong VI. A scoping review on the prevalence and determinants of post-traumatic stress disorder among military personnel and firefighters: Implications for public policy and practice. Int. J. Environ. Res. Public Health. 2022;19(3):1565. Arora D, Belsiyal CX, Rawat VS. Prevalence and determinants of posttraumatic stress disorder and depression among survivors of motor vehicle accidents from a hilly Indian state. Indian J. Psychiatry. 2021;63(3):250-7. Daddah D, Glele Ahanhanzo Y, Kpozehouen A, Hounkpe Dos Santos B, Ouendo E-M, Levêque A. Prevalence and risk factors of post-traumatic stress disorder in survivors of a cohort of road accident victims in Benin: results of a 12-month cross-sectional study. J. Multidiscip. Healthc. 2022:719-31. Lee DY, Kim E, Choi MH. Technical and clinical aspects of cortisol as a biochemical marker of chronic stress. BMB Rep. 2015;48(4):209. Steudte S, Kirschbaum C, Gao W, Alexander N, Schönfeld S, Hoyer J, et al. Hair cortisol as a biomarker of traumatization in healthy individuals and posttraumatic stress disorder patients. Biol. Psychiatry. 2013;74(9):639-46. Laufer S, Engel S, Lupien S, Knaevelsrud C, Schumacher S. The Cortisol Assessment List (CoAL) A tool to systematically document and evaluate cortisol assessment in blood, urine and saliva Compr. Psychoneuroendocrinol. 2022;9:100108. Russell E, Kirschbaum C, Laudenslager ML, Stalder T, de Rijke Y, van Rossum EF, et al. Toward standardization of hair cortisol measurement: results of the first international interlaboratory round robin. Ther. Drug Monit. 2015;37(1):71-5. Pacella ML, Hruska B, Steudte-Schmiedgen S, George RL, Delahanty DL. The utility of hair cortisol concentrations in the prediction of PTSD symptoms following traumatic physical injury. Soc. Sci. Med. 2017;175:228-34. Alghnam S, Alyabsi M, Aburas A, Alqahtani T, Bajowaiber M, Alghamdi A, et al. Predictors of seatbelt use among Saudi adults: results from the National Biobank Project. Front. Public Health. 2020;8:579071. Taranov A, Puchkova A, Slominsky P, Tupitsyna T, Dementiyenko V, Dorokhov V. Associations between chronotype, road accidents and polymorphisms in genes linked with biological clock and dopaminergic system. Zhurnal nevrologii i psikhiatrii imeni SS Korsakova. 2017;117(4. Vyp. 2):28-33. Blatt RJ. Banking biological collections: data warehousing, data mining, and data dilemmas in genomics and global health policy. Public Health Genomics. 2001;3(4):204-11. Linsen L, Van Landuyt K, Ectors N. Automated sample storage in biobanking to enhance translational research: the bumpy road to implementation. Front. Med. 2020;6:309. Malek A, Dadashzadeh H, Poorafkary N. Ranking of stressful life events in general population of Tabriz–Iran. Med. J. Tabriz Univ. Med. Sci. Health Serv. 2009;30(4):73-80. Dadfar M, Kalibatseva Z, Lester D. Reliability and validity of the Farsi version of the Patient Health Questionnaire-9 (PHQ-9) with Iranian psychiatric outpatients. Trends Psychiatry Psychother. 2018;40(2):144-51. Wang X, Zuo Y, Jiang H, Yang L. Relationship between the incidence of road traffic accidents, psychological characteristics, and genotype in bus drivers in a Chinese population. Medical science monitor: Int J Exp Clin Res. 2018;24:5566. Golestani M, Razzaghi A, Rezaei M, Vahedi L, Pourasghar F, Farahbakhsh M, et al. PERSIAN traffic safety and health cohort: a population-based precrash cohort study. BMJ Open. 2024;14(2):e080720. Sadeghi-Bazargani H, Shahedifar N, Somi MH, Poustchi H, Bazargan-Hejazi S, Jafarabadi MA, et al. PERSIAN Traffic Safety and Health Cohort: a study protocol on postcrash mental and physical health consequences. Inj Prev. 2022;28(3):269-79. Table Table 1. Responsibilities of Biobank Key Members Role Responsibilities Program Principal Coordination with all committees, experts, and the software team; organizing intergroup meetings; financial management; overseeing the scientific and operational processes of the program; reporting; and article writing. Executive Committee Procuring necessary materials and equipment; duplicating and completing questionnaires; blood collection; preparing hair and blood samples; delivering samples to centers; serum preparation; biobank storage; sample control; coding; data entry into the software; reporting; and article writing. Software and Quality Control Committee Developing software programs; quality control; assisting with data outputs; maintaining continuous communication between the software team and the executive secretary for accurate data entry and quality control; reporting; and article writing. Safety and Sample/Information Access Committee Sample storage; safeguarding patient information; adhering to ethical principles in sample preservation; communicating with other committees; and providing samples and data to researchers based on the ethical charter of the program. Scientific Committee Reporting; article writing; and defining and developing scientific projects. Table 2. Policies of Biobank Program Cost-effectiveness of the program Collection of samples in a manner that allows a broad range of possible measurements Minimization of time for collection, processing, transportation, and archiving of samples Prevention of potential sample degradation during transportation and archiving Quality assurance (preventing errors and variability) and quality control methods (detecting errors and variability when they occur) Ensuring the security of samples and data Management of sample and data usage Management of sample and data sharing Standardization and centralization of processing at a dedicated site Staff training on agreed-upon and approved operational procedures Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":78581,"visible":true,"origin":"","legend":"\u003cp\u003eBiobank study algorithm for traffic accident victims\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8409982/v1/552a3c588e5ec42967bd3eab.jpg"},{"id":100396859,"identity":"c05c5b68-9c00-4cf1-876e-b2a4ce35ff71","added_by":"auto","created_at":"2026-01-16 11:41:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42099,"visible":true,"origin":"","legend":"\u003cp\u003eProcedure of hair Sampling\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8409982/v1/daca40058f0960fa78b6dd60.jpg"},{"id":100396952,"identity":"a2b6b544-09bd-42d5-a2b8-76d2ff408795","added_by":"auto","created_at":"2026-01-16 11:41:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47563,"visible":true,"origin":"","legend":"\u003cp\u003eStorage area for blood samples in the central biobank\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8409982/v1/416828eb6148d476e0d49c2e.jpg"},{"id":103395899,"identity":"7710a9d2-040a-4ee3-a1a5-8cc9ef01e13e","added_by":"auto","created_at":"2026-02-25 08:43:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":965376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8409982/v1/26060ed9-7cb8-446c-8746-7401c2c7f341.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishing a biobank for road traffic accident victims in Iran as a cohort study of stress related biomarkers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, the increasing mortality and injuries due to road traffic accidents have remained one of the leading causes of disability and a major public health concern.\u003csup\u003e(1)\u003c/sup\u003e Annually, road traffic accidents result in injuries to 20 to 50\u0026nbsp;million individuals worldwide, with approximately 1.19\u0026nbsp;million fatalities.\u003csup\u003e(2)\u003c/sup\u003e The fatality rate due to traffic injuries has decreased in developed countries, whereas it continues to increase in low- and middle-income countries, such that nearly 90% of global deaths from traffic injuries occur in these regions.\u003csup\u003e(3)\u003c/sup\u003e Iran is among the countries with the highest rates of traffic accident fatality and disability worldwide.\u003csup\u003e(4)\u003c/sup\u003e In addition to causing death and disability, traffic accidents can also lead to a variety of social, economic, and psychological consequences.\u003csup\u003e(5)\u003c/sup\u003e Post-traumatic stress disorder (PTSD) is considered one of the most common mental health consequences of traffic accidents.\u003csup\u003e(6)\u003c/sup\u003e Epidemiological studies indicate that not all individuals exposed to the same traumatic event will develop PTSD; rather, individual vulnerability plays a significant role in this regard. This vulnerability can be either genetic or acquired after birth.\u003csup\u003e(7)\u003c/sup\u003e Therefore, identifying the factors that contribute to traffic accidents as well as PTSD is of great importance. Among these factors, environmental influences, demographic characteristics, and individual factors such as stress can be highlighted.\u003csup\u003e(8,9)\u003c/sup\u003e Stress is one of the risk factors for chronic diseases that can lead to both physical and psychological issues, and manifests in two forms: acute and chronic. Acute stress, through its impact on the hypothalamus and the transmission of nerve impulses to the central part of the adrenal gland, results in the secretion of catecholamine hormones, that is, epinephrine and norepinephrine.\u003csup\u003e(10)\u003c/sup\u003e In contrast, in chronic stress, the hypothalamic-pituitary-adrenal (HPA) axis is activated, ultimately yielding the secretion of cortisol hormone from the adrenal cortex. This hormone has long-term effects and is involved in the pathophysiology of a wide range of physical and psychological problems. Cortisol has recently become a key focus in psychoneuroendocrinology research.\u003csup\u003e(11)\u003c/sup\u003e Although it can be detected in blood, urine, and saliva, its levels fluctuate significantly over a 24-hour period \u003csup\u003e(12)\u003c/sup\u003e. In contrast, hair provides a reliable medium for assessing cortisol levels \u003csup\u003e(11)\u003c/sup\u003e, as cortisol enters hair primarily through passive diffusion. Given that hair grows at a relatively constant rate of 1 centimeter per month, each centimeter reflects roughly one month of cortisol production, thereby mitigating the issue of cortisol fluctuations \u003csup\u003e(13)\u003c/sup\u003e. Moreover, by segmenting hair samples into different sections and analyzing them separately, a retrospective timeline of exposure to substances and the onset of disease can be determined \u003csup\u003e(14)\u003c/sup\u003e. Accurate identification of the factors influencing traffic injuries and PTSD requires the standard collection of biological samples and the analysis of a large volume of biological data. Biological samples are valuable and non-renewable resources, particularly when accompanied by follow-up and clinical care \u003csup\u003e(15)\u003c/sup\u003e. Each biological sample should be considered not only as a small genetic and molecular resource for research programs but also as a database for advanced patient treatments based on personalized medicine. Personalized medicine, based on the genetic characteristics of each individual, focuses on treating people \u003csup\u003e(16)\u003c/sup\u003e. One of the key tools for research on personalized medical is the study of human biological samples, including hair, nails, DNA, and serum. The establishment of biobanks facilitates access to biological samples \u003csup\u003e(17)\u003c/sup\u003e. Biobanks consist of tissues, organs, plasma, saliva, DNA, RNA, proteins, living cells, genetic material from living organisms, hereditary information, and functional data related to biological systems, all preserved for research purposes over an extended period. Thus, biobanks act as the cornerstone of personalized medicine \u003csup\u003e(18)\u003c/sup\u003e. Their benefits are realized when they are established with careful attention to project objectives, sample quality control, high-quality data generation, data integrity, security, confidentiality, and the management and sharing of samples\u0026mdash;all of which are critically important \u003csup\u003e(17)\u003c/sup\u003e. Considering the role of chronic stress in occurrence of traffic accidents and their severe consequences, both of which impose significant functional and financial burdens, research on biological samples is crucial to identify individuals at risk for such accidents and to prevent post-crash complications. Therefore, given the lack of previous efforts to collect biological samples from traffic accident victims, the establishment of a biobank for these individuals in Iran is indispensable. This study aims to design and create a protocol for a biobank of road traffic victims.\u003c/p\u003e"},{"header":"Methods and analysis","content":"\u003cp\u003eThis prospective cohort study will be conducted over two years on individuals hospitalized following traffic accidents at Imam Reza hospital (as educational and referral hospital) in Tabriz University of medical Sciences/Iran. The study is aligned with the Tabriz University of Medical Sciences' Road Traffic Injury Registry Program and the Persian Traffic Health and Safety Cohort Program, contributing to the development of both initiatives. Figure 1 illustrates the overall study algorithm. The methodology of the present study is structured in two sections: the establishment and implementation of the biobank.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEstablishment of the biobank program for the traffic accident victims\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe first step in establishing and implementing the biobank is its conceptualization, aimed at identifying and defining the foundational structures of the program. For the conceptualization of the traffic accident victim biobank, an organizational structure for the program was first established. Accordingly, the program is implemented under the supervision of the Vice Chancellor for Research and Technology at Tabriz University of Medical Sciences, within the RTIRC. The program comprises committees such as the Executive Committee, Scientific Committee, Software and Quality Control Committee, and the Safety and Sample/Information Access Committee. The roles of the program’s key members are detailed in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1. \u003cstrong\u003eResponsibilities of Biobank Key Members\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, the management of the biobank's operational activities, the process for data sharing, and the policies for utilizing the biobank's resources for traffic accident victims were defined. The ten key policies are outlined in Table 2. Data sharing will occur through the website of the RTIRC, where an announcement will be issued regarding the use of samples and data, in accordance with the conditions and regulations specified in the ethical guidelines. The ownership of these samples is held by the RTIRC at Tabriz University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003eTable 2. Policies of Biobank Program\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the necessary physical infrastructure for implementing the program was determined and prepared. In this regard, Imam Reza hospital was designated for the first phase that is sample collection from individuals admitted to this hospital following a traffic accident, the HTSC for the follow-up phase that is re-sampling, psychiatric consultation, and other necessary assessments, and the central biobank of Tabriz University of Medical Sciences for storing blood samples.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImplementation of the biobank program for the traffic accident victims\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe program will be executed in two phases. In the first phase, individuals hospitalized due to traffic accidents at Imam Reza Educational and Medical Center in Tabriz will be identified by biobank experts stationed at the center. After assessing inclusion and exclusion criteria, their names and details will be recorded in the Traffic Accident Registration System. Subsequently, with ethical considerations in place, participants will complete demographic questionnaires, hair condition forms, and questionnaires on stress-inducing life events. Finally, biological samples (hair and blood) will be collected in accordance with prior instructions and training.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo follow up and screen the traffic accident victims, biobank experts at the RTIRC will make phone calls two months after the road traffic. During these calls, follow-up forms will be completed. If a participant scores PTSD ≥ 3 or PHQ ≥ 5, they will be invited to the HTSC for further evaluation. At the clinic, after completing the relevant questionnaires, participants will undergo a driving test using a simulator, an assessment via the Vienna Test System, and a consultation with a psychiatrist. If victims meet the criteria for PTSD as outlined in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition), they will be classified as PTSD-positive. Finally, biological samples (hair and blood) will be collected again from participants, both PTSD-positive and PTSD-negative.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParticipants and sample size\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe participants in this study include individuals hospitalized at Imam Reza Hospital due to traffic accidents, whose information is recorded in the Iranian Integrated Road Traffic Injury Registry (IIRTIR) within Region two of the National Healthcare Map. Individuals eligible for inclusion in the study are those aged over 18 years who express willingness to participate in the research. However, several exclusion criteria apply: individuals unwilling to cooperate, those with hair shorter than 3 centimeters, those who have used systemic or topical steroids on the scalp within the past three months, and individuals who are currently pregnant or have been pregnant within the past three months. Additionally, individuals with severe illnesses such as cancer, HIV, or autoimmune disorders, those who have abused alcohol or substances within the past three months, and individuals with current major depressive disorder (MDD) accompanied by psychotic symptoms and suicidal thoughts are excluded. Participants with bipolar disorder or suicidal thoughts within the past three months are also excluded. The sample size is based on a study by Russell \u003csup\u003e(15)\u003c/sup\u003e, with 20 participants estimated per month. Given the planned two-year study duration, a total of 600 participants (400 cases in the initial phase and 200 cases in the follow-up phase) are expected. Participants will be selected using a non-random convenience sampling method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData and biological sample collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, data such as demographic characteristics, hair-related information, and details of life stressor events will be collected through questionnaires. Hair and blood samples will be collected by biobank experts at both Imam Reza hospital and the RTIRC. Moreover, psychiatric examinations will be conducted at the RTIRC by a psychiatrist through interviews with the patients. The details of each data collection method are described below.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData collection via questionnaires\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter obtaining informed consent and clearly explaining the study objectives to the participants, and considering the inclusion and exclusion criteria, the questionnaires will be filled out at Imam Reza hospital during the first phase, and at the RTIRC, during the two-month follow-up screening phase. The questionnaires will be stored at the RTIRC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuestionnaire on demographic information and hair status of victims\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare the questionnaire, methods such as literature review, examining biobank registration centers in other countries, conducting a pilot study, and brainstorming were utilized. Eventually, a semi-structured questionnaire was designed. This questionnaire includes a series of questions in three areas: demographic information, hair status, and previous accidents of the victims. The questions cover aspects such as age, gender, education, marital status, weight, height, alcohol consumption, cigarette smoking, contraceptive use, menopausal status, pregnancy status, breastfeeding status, underlying medical conditions, regular use of medications, hair dyeing, other hair treatments such as perming, hair extensions, or straightening, frequency of hair washing within a week, hours of direct sun exposure to the hair in a day, number of severe accidents experienced, hair cortisol levels, and timing of sample collection. The validity of this questionnaire was confirmed through content validity and face validity methods.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuestionnaire on stress-inducing life events of victims\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis questionnaire pertains to the ranking of stress-inducinglife events and measures the amount of stress an individual has experienced over the course of a year. The Holmes and Rahe Stress Scale includes 43 life events, with each assigned a numerical rating. Each event is considered a unit of life change with a different weight in generating stress. If an event has occurred more than once for the individual, the corresponding score is doubled before summing the total to derive the final score. For a score between 11 and 150, there is a very low to moderate likelihood that the individual will become ill due to stress in the near future. For a score between 150 and 299, the likelihood of illness due to stress increases to moderate or high. For scores between 300 and 600, there is a high to very high probability that the individual will become ill due to stress. Maleki et al. conducted a cross-sectional descriptive study on the population of Tabriz. Considering cultural and social differences, they modified the Holmes-Rahe Stress Questionnaire and developed a 50-item questionnaire assessing stress-inducing events \u003csup\u003e(19)\u003c/sup\u003e. The evaluation of participants will be based on this adapted questionnaire, focusing on stressful events experienced over the past year.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe database scheme\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study uses a relational database developed in Microsoft Access to manage and organize data collected from participants in a prospective cohort study on chronic stress and its role in accidents and PTSD. The database scheme is designed to facilitate the integration of multiple data types, including demographic information and Stressful life events. The database consists of four primary tables linked by a unique identifier, the National ID Number. This identifier is used to ensure that data from various sources can be accurately combined for each individual.\u003c/p\u003e\n\u003cp\u003eThe four tables in the database are:\u003c/p\u003e\n\u003cp\u003e1. Demographic information, hair status, and medication consumption: This table stores participant demographics (e.g., age, gender, occupation, and etc.) and details on hair status (e.g., hair washing frequency, hair dying) and medication consumption.\u003c/p\u003e\n\u003cp\u003e2. Stressful life events experienced by the individuals during the last three spans: This table records stressful life events and the scores assigned to each question participants experienced during the last three spans i.e. three months, six months and 12 months including events like job loss, family death, financial problems, and etc.\u003c/p\u003e\n\u003cp\u003eDue to the field limitation in Microsoft Access (i.e. less than 255 fields per table), the second and third tables, related to stressful life events, contain the remaining questions to accommodate all questions appropriately. Consequently, the data from stressful life events is spread across several tables, guaranteeing that each participant’s data is registered in full.\u003c/p\u003e\n\u003cp\u003eEach table includes a unique AutoNumber field named ID, which serves as the primary key for data entry and ensures accurate linking between tables. Data types of the fields in these tables are \"Short Text,\" \"Yes/No,\" and \"Number\" corresponding to responses on the questionnaires.\u003c/p\u003e\n\u003cp\u003eData sources include structured questionnaires (on demographics and stressful life events), biological samples (hair and blood for measuring cortisol levels), and hospital records from Imam Reza Hospital, Tabriz registered in the IIRTIRS. Data entry is carried out by trained research staff, following strict protocols to assure accuracy and consistency. The database supports data integration from the data sources, providing a comprehensive view of participant experiences and stress biomarkers.\u003c/p\u003e\n\u003cp\u003eQuality control includes automated data validation checks, regular audits, and the use of data-entry protocols to minimize human error. Data is securely stored, and access is restricted to authorized personnel to maintain confidentiality and integrity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe PTSD diagnostic questionnaire\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis questionnaire is a follow-up form for traffic accident survivors aged 18 and above, focusing on events within the past month. It is to be completed via phone and includes five yes/no questions, with the total score determined by summing responses. Higher scores indicate greater severity of the disorder. Individuals scoring three or higher are referred to the THSC for further evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe PHQ-9 questionnaire\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis questionnaire is a widely used tool over the past two decades for assessing depression. It consists of ten questions, with the first nine corresponding to DSM-5 criteria for major depressive disorder, such as sadness and hopelessness, loss of interest or pleasure, sleep disorders, fatigue or low energy, appetite changes, feelings of worthlessness or guilt, agitation, difficulty in concentrating, and suicidal thoughts\u003c/p\u003e\n\u003cp\u003eIn the last question, the overall functioning of the individual is assessed. Each question has four response options: \"not at all,\" \"several days,\" \"more than half the days,\" and \"nearly every day,\" scored from 0 to 3, respectively. To diagnose depression, at least five questions must be marked as \"more than half the days\" or \"nearly every day.\" To calculate the total score of the PHQ-9 questionnaire, the scores for each question are summed up, and a new variable is generated. If the total score exceeds four, it signifies depression. The reliability of this questionnaire with a Cronbach alpha of 88% and content validity of 74% has been confirmed in the study by Dadfar et al.\u003csup\u003e(20)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData collection through behavior measurement devices\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe Vienna Test System (VTS)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVTS is a computerized tool designed for psychological, neurological, and transportation evaluations, offering high objectivity and precision, including factors that traditional pen-and-paper tests cannot measure. The indigenous driving performance assessment device, developed by the Traffic Research Center at Tabriz University of Medical Sciences, is based on data from Iranian participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBefore the main tests, individuals perform practice exercises to familiarize themselves with the system. Tests in this study include assessments for peripheral vision, reaction time, and other relevant measures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDriving test in a simulator\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Sahand Driving Simulator was developed in 2018 by the Traffic Research Center at Tabriz University of Medical Sciences. This simulator consists of a real cabin based on a Peugeot 405, one of the most commonly used cars in Iran, and three 43-inch monitors. The software used in the simulator is openDS, an open-source program that has been enhanced to meet the research needs of the RTIRC. The scenario used in this study includes driving in three different settings: rural, urban, and highway environments. The total driving time for the scenario ranges from 23 to 30 minutes. Notably, all drivers first complete a five-minute practice scenario to make themselves familiar with the simulator before starting the main scenario.\u003c/p\u003e\n\u003cp\u003eThe simulator assesses various driving functions designed to evaluate driving abilities related to response to unexpected events, collision avoidance behavior, total brake reaction time, simulator performance index, driving performance measures, and continuous time to collision. These skills are evaluated through the scenarios, providing an assessment of each individual’s driving performance. The validity and reliability of the driving simulator have been confirmed by the RTIRC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCollection of biological samples\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce informed consent is obtained and the objectives are clearly explained to the participants, hair and blood samples will be collected. The sample collection will be performed by trained experts, taking into account the inclusion and exclusion criteria as well as ethical considerations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProcedure of hair sample collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWearing gloves and using sterilized scissors, biobank experts will collect approximately 100 hair strands from the posterior region of the scalp, with each hair strand being about the thickness of a pencil and 3 cm in length. After identifying the proximal end of the hair, it will be placed inside a 5*5 cm piece of thick aluminum foil (Figure 2). The foil will then be placed inside a 5*7 cm ziplock bag along with 10 to 15 desiccants or silica gel packets. The ziplock bags will be stored in Frozen (-20°C). Each ziplock bag will be labeled with a unique code assigned to each patient, which must match the codes both on the questionnaires and in the IIRTIRS. Finally, the hair samples must be stored in a dry and dark environment, away from direct sunlight, at the HTSC of the RTIRC. Hair samples should not be stored in a refrigerator or freezer. It is worth mentioning that scalp hair is the preferred sample; however, if scalp hair is not available, particularly in men, hair samples may be collected from other parts of the body, such as the arms, chest, underarms, beard, and legs. The specific area from which the hair was collected must be recorded in the corresponding section of the hair archiving list under \"Notes.\" The collection of hair samples requires careful attention to the donor's privacy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 2. Procedure of hair Sampling\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProcedure of blood sampling\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples will be collected by a trained individual wearing gloves. A total of 10 cc of venous blood will be drawn into ethylenediaminetetraacetic acid (EDTA)\u0026nbsp;anticoagulant tubes. The tubes will be gently inverted to prevent the formation of blood clots. After collecting the blood sample in EDTA tubes, it is centrifuged, then the white blood cells (buffy coat layer) are extracted. Immediately after separation, they are aliquoted and placed in special cryovial tubes. Protective solutions such as TE buffer are added to help stabilize the DNA, and then the samples are frozen at -80°C, where DNA will be extracted from the blood as soon as possible (based on approved projects) (Figure 3). The collection of venous blood samples is contingent on sufficient funding, as well as the collaboration of Imam Reza hospital and the central biobank. Each EDTA anticoagulant tube will be labeled with a unique code assigned to each patient. These codes must also match the codes both on the questionnaires and in the IIRTIRS.\u003c/p\u003e\n\u003cp\u003eFigure 3. Storage area for blood samples in the central biobank\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePsychiatric examinations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePsychiatric examinations will be conducted during the follow-up phase by a psychiatrist for participants who have visited the HTSC. Following the examinations, the participants will be classified into three groups: the first group consists of participants whose post-traumatic stress disorder (PTSD) is positive in both the screening and the psychiatrist's examination. The second group includes participants whose PTSD is positive in the screening but negative in the psychiatrist's examination. The third group comprises participants whose PTSD is negative in both the screening and the psychiatrist's examination. Subsequently, the results of the questionnaires and hair cortisol levels will be measured and compared among the three groups. The measurement of hair cortisol will be done using the ELISA method and Liquid chromatography–mass spectrometry\u0026nbsp;(LC–MS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuality control\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral approaches have been considered by the central study team to ensure and maintain the quality of the follow-up implementation process, which include:\u003c/p\u003e\n\u003cp\u003e1. Evaluation of the quality of follow-up implementation on the Central Biobank Quality Assurance and Control Committee’s behalf \u0026nbsp;at six-month intervals.\u003c/p\u003e\n\u003cp\u003e2. Monthly review of the accuracy of completed follow-up forms and data cleaning by examining forms that have been recorded as incomplete, incorrect, or duplicate.\u003c/p\u003e\n\u003cp\u003e3. Monthly verification of the accuracy of form completion for each field by conducting follow-up phone calls with at least 10% of participants in each field, selected randomly, by the Biobank Quality Control Committee.\u003c/p\u003e\n\u003cp\u003e4. Completion of self-assessment forms by interviewers and other follow-up team members at one-month intervals.\u003c/p\u003e\n\u003cp\u003e5. Monthly evaluation of the required equipment and human resources.\u003c/p\u003e\n\u003cp\u003e6. Assessment of the quality of blood sample preparation and storage, and other biological samples.\u003c/p\u003e\n\u003cp\u003e7. Review of the quality and accuracy of the work performed by the biobank experts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaintenance of ultra-low temperature freezers (-80\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003edegrees Celsius\u003cem\u003e) in the biobank\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltra-low temperature (ULT) freezers store contents at −80°C\u0026nbsp;and are among the most critical equipment in a biobank laboratory. The primary principles for extending the lifespan and ensuring the cooling system's efficiency, as well as preserving sample quality, include installing digital temperature monitoring systems and recording daily temperature fluctuations using dedicated software with notification capabilities. Temperature fluctuations inside the freezer exceeding ±1.5°C are undesirable.\u0026nbsp;The freezer should be cleaned every six months and undergo an annual freeze and defrost cycle, during which the freezer is emptied, powered off, cleaned, and restarted. In addition, the frost on the freezer door should be removed weekly, and the door gasket should be inspected regularly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eReporting\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll team members must promptly deliver critical and significant reports to the biobank manager (via phone). The Quality Control Committee is required to submit a monthly report of the quality control assessment results to the biobank manager. Interviewers and other members of the implementation team must report any challenges and limitations in executing the guidelines to the biobank manager.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRevision, Rescheduling, and Retraining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the quality control results, the need for retraining follow-up team members should be considered. Retraining will be conducted by the biobank manager. It is also important to note that the number of cohort participants decreases over time due to deaths or withdrawal from the study, highlighting the necessity for the biobank manager to adjust the schedule accordingly. The biobank manager is responsible for reviewing the necessary equipment and human resources, identifying any shortages or issues, and reporting them to the central team at monthly intervals or, if needed, immediately for appropriate action. If the implementation of this protocol becomes unfeasible or fails to meet the study's objectives, the follow-up activities should be halted, and a new protocol should be developed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBiobank’s database management system\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, the information obtained from the victims will be entered into the IIRTIRS (International Institute for Road Traffic Incident Reporting System). Once the questionnaires are completed, the data will be entered into the database management system, which links questionnaire responses with biological sample data using a unique identifier (e.g., national code) assigned to each victim. This guarantees the proper tracking and management of each participant’s data across all records. The registry experts at the RTIRC will conduct the data quality control. Additionally, one data committee member will act as a liaison between the database management system and the user.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMeasurement of cortisol concentration in hair\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo measure cortisol concentration in hair, two methods will be utilized: ELISA and chromatography, based on the study by Russell et al. \u003csup\u003e(13)\u003c/sup\u003e. The summarized steps for the ELISA method, according to Kirschbaum et al., are as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHair washing, hair powdering, solvent extraction, incubation, centrifugation during extraction, evaporation, reconstitution with solvent, vortexing, use of ELISA and consideration of cross-reactivity, centrifugation during ELISA and consideration of wavelength when measuring cortisol.\u003c/p\u003e\n\u003cp\u003eIn the ELISA method, ALPCO's of ELISA kits (New Hampshire, USA) will be used. Liquid chromatography-mass spectrometry (LC-MS/MS), or the chromatography method, will be performed at Faculty of Pharmacy/Tabriz University of Medical Sciences/Iran. DNA extraction and genetic analysis of blood samples will also be carried out based on similar projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is designed to collect data on road accidentvictims and serves as an infrastructure research project. The information gathered in this study will be utilized in future research projects based on their specific objectives.In general, the study will assess demographic data, trauma-related questionnaires, stress levels, the presence of PTSD, and hair cortisol levels across three groups. To compare qualitative data, such as gender and the presence or absence of PTSD among the three groups, the Chi-square test will be used. For comparing quantitative data, such as stress questionnaire scores and hair cortisol levels among the three groups, analysis of variance (ANOVA) will be used.The Pearson correlation test will be applied to assess the correlation of hair cortisol levels between the ELISA and chromatography methods. Logistic regression analysis will then be used to predict the odds of developing PTSD. P-value\u0026lt; 0.05 will be considered statistically significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRoad traffic accidents and their consequences are among the leading causes of mortality and disability across various age and gender groups in low- and middle-income countries \u003csup\u003e(3)\u003c/sup\u003e. Genetic factors are a key determinant of individuals' vulnerability to traffic accidents and the complications following them \u003csup\u003e(7)\u003c/sup\u003e. Hence, research aimed at uncovering the genetic mechanisms contributing to traffic accidents and their consequences is of critical importance. Biobanks serve as a reliable infrastructure for collecting and preserving biological samples. The findings derived from these biobanks can provide valuable evidence for understanding the genetic factors influencing the risks and outcomes of traffic accidents, enabling researchers worldwide to make valid and evidence-based decisions \u003csup\u003e(17,21)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study provides detailed information on the design and establishment of the biobank for road accidentvictims, a pioneering initiative in the country. The biobank will be launched and executed by the RTIRC, which has been officially designated as the national reference hub for traffic and health by the relevant authorities. The purpose of this biobank is to collect, store, and analyze biological samples, such as hair and blood, from individuals hospitalized due to traffic accidents. The RTIRC, recognized as the site of the country\u0026rsquo;s first traffic cohort \u003csup\u003e(22, 23)\u003c/sup\u003e and a WHO Collaborating Center, is a leading institution in traffic safety in Iran. Therefore, during the implementation of the biobank, it can leverage its previous experiences and international collaborations to promote the program. As part of this project, protocols and guidelines for establishing the biobank will first be prepared, and data collection will be carried out accordingly. Subsequently, universities nationwide will be notified about the existing data to encourage their participation in future biobank-related projects across various disciplines. International researchers will also be invited to collaborate, leveraging their expertise for the development and implementation of upcoming biobank projects. To maintain and enhance the scientific and operational quality of the biobank, continuous updates to protocols, ongoing staff training, expansion of sampling centers, collaboration with international biobank development centers, partnerships with other national universities to establish related biobanks, and securing sustainable financial resources may be required over time. Furthermore, a comprehensive network of experts in medical genetics, traffic injuries, psychiatry, chemistry, pharmacy, epidemiology, and biostatistics is essential. Adherence to ethical and legal guidelines, strong software infrastructure, advanced hardware equipment, and adequate human resources are also critical for the optimal operation of the biobank. Therefore, the biobank program must receive full support from national authorities, as well as governmental and non-governmental organizations in these aspects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDue to the impact of chronic stress on the occurrence of road accidents, and the severe complications that can follow such accidents, including PTSD, which imposes a significant functional and financial burden, the current research aimed at identifying individuals at risk of road accidents (including those experiencing chronic stress) and preventing the complications that may arise after road accidents (such as PTSD) appears to be important. Future studies will investigate hair cortisol as a means to reduce the incidence of road accidents and PTSD. Moreover, efforts will be made in this research to compare different methods of measuring hair cortisol, with the goal of identifying a method that is more affordable, more accessible, and highly sensitive.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStrengths and limitations of this study\u003c/b\u003e: The study's strengths are as follows: The study was conducted on a cohort of road accident victims who were admitted to the hospital. Demographic information, hair characteristics, and stressful life events were recorded for the participants. Blood and hair samples were also prepared for analysis. The study's limitations are as follows: The majority of the women's hair samples were dyed with the second color. Due to a lack of adequate facilities, it was not possible to obtain RNA samples.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTraffic Injury Management and Prevention Research Center\u003c/p\u003e\n\u003cp\u003eRoad traffic injury research center (RTIRC)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHealth and Traffic Safety Clinic\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(HTSC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTraffic and Health Reference Hub\u003c/p\u003e\n\u003cp\u003eIranian Integrated Road Traffic Injury Registry (IIRTIR)\u003c/p\u003e\n\u003cp\u003ePost-traumatic stress disorder (PTSD)\u003c/p\u003e\n\u003cp\u003eHypothalamic-pituitary-adrenal (HPA)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe present study has been approved by the Regional Ethics Committee of Tabriz University of Medical Sciences under ethics code IR.TBZMED.REC.1401.950. Unlike other research projects, the specific type of tests to be conducted on biological samples and their potential results are not predetermined in a biobank. Therefore, ethical considerations the Declaration of Helsinki and its later are of utmost importance in biobanks. The informed consent form for the biobank of the RTIRC has been developed based on the American Society of Human Genetics guidelines regarding the storage of biological samples and data. Informed consent was obtained from all the individual participants for participation in this study. After receiving necessary explanations and addressing any potential ambiguities, individuals voluntarily and informedly sign the consent form. These signed informed consent forms are securely stored in the biobank unit of the RTIRC at the Tabriz University of Medical Sciences. The consent form explicitly states that the study is just conducted for research purposes and does not provide any diagnostic commitments. However, if any findings obtained during the research process are deemed significant for the diagnosis, disease progression, or treatment of participants, the individuals will be informed.\u003c/p\u003e\n\u003cp\u003eParticipation in the study is entirely voluntary, and participants may withdraw at any time. The questionnaire data and biological samples of patients will be kept strictly confidential. No names will be used on questionnaires or biological samples. The data entered into the computer system will also remain confidential, and the computer will be equipped with a security system to prevent unauthorized access to patient information. Data will be anonymized by use of codes and kept confidential. Confidentiality will be maintained even for other researchers who may use the samples and data in future studies. All data will be handled in accordance with Iranian data protection laws, guaranteeing their confidentiality. The use of biobank samples by other researchers for future studies will be conducted in accordance with the ethical charter established by the center. These researchers are required to comply with the principles of data use and confidentiality signing an agreement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eParticipation in the study is entirely voluntary, and participants may withdraw at any time. The questionnaire data and biological samples of patients will be kept strictly confidential. No names will be used on questionnaires or biological samples. The data entered into the computer system will also remain confidential, and the computer will be equipped with a security system to prevent unauthorized access to patient information. Data will be anonymized by use of codes and kept confidential. Confidentiality will be maintained even for other researchers who may use the samples and data in future studies. All data will be handled in accordance with Iranian data protection laws, guaranteeing their confidentiality. The use of biobank samples by other researchers for future studies will be conducted in accordance with the ethical charter established by the center. These researchers are required to comply with the principles of data use and confidentiality signing an agreement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u0026nbsp;\u003c/strong\u003eNot applicable as no information or images of the participants are present in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003eData are available from the authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eRoad Traffic Injury Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. (Grant Number: 70347).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVL:\u0026nbsp;\u003c/strong\u003eContributions to the conception and design of the work, control and interpretation of data, writing the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFM\u003c/strong\u003e: Examination of the survivors, interpretation of data, revision of the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNA\u003c/strong\u003e: Design and creation of the database used in the study, control data, writing the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSH\u003c/strong\u003e: Contributions to the conception and design of the work, interpretation of data, revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors\u0026nbsp;would like to thank all the Road Traffic Injury of Registry Team Members for collaboration in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOrganization WH. Global status report on road safety 2018: World Health Organization; 2019.\u003c/li\u003e\n\u003cli\u003eOrganization WH. Global status report on road safety 2023: summary: World Health Organization; 2023.\u003c/li\u003e\n\u003cli\u003ePerel P, Ker K, Ivers R, Blackhall K. Road safety in low-and middle-income countries: a neglected research area. Inj Prev. 2007;13(4):227-.\u003c/li\u003e\n\u003cli\u003eMehryar HR, Alizadeh N, Rostampour F, Paryab S, Bab S, Garkaz O. Causes of road accidents in northwestern iran in the period 2010\u0026ndash;2018. Int. Arch. Health Sci. 2022;9(1):14-9.\u003c/li\u003e\n\u003cli\u003eBoelen PA, Eisma MC, de Keijser J, Lenferink LI. Traumatic stress, depression, and non-bereavement grief following non-fatal traffic accidents: Symptom patterns and correlates. PLoS One. 2022;17(2):e0264497.\u003c/li\u003e\n\u003cli\u003evan den Heuvel LL, Stalder T, du Plessis S, Suliman S, Kirschbaum C, Seedat S. Hair cortisol levels in posttraumatic stress disorder and metabolic syndrome. Stress. 2020;23(5):577-89.\u003c/li\u003e\n\u003cli\u003eObuobi-Donkor G, Oluwasina F, Nkire N, Agyapong VI. A scoping review on the prevalence and determinants of post-traumatic stress disorder among military personnel and firefighters: Implications for public policy and practice. Int. J. Environ. Res. Public Health. 2022;19(3):1565.\u003c/li\u003e\n\u003cli\u003eArora D, Belsiyal CX, Rawat VS. Prevalence and determinants of posttraumatic stress disorder and depression among survivors of motor vehicle accidents from a hilly Indian state. Indian J. Psychiatry. 2021;63(3):250-7.\u003c/li\u003e\n\u003cli\u003eDaddah D, Glele Ahanhanzo Y, Kpozehouen A, Hounkpe Dos Santos B, Ouendo E-M, Lev\u0026ecirc;que A. Prevalence and risk factors of post-traumatic stress disorder in survivors of a cohort of road accident victims in Benin: results of a 12-month cross-sectional study. J. Multidiscip. Healthc. 2022:719-31.\u003c/li\u003e\n\u003cli\u003eLee DY, Kim E, Choi MH. Technical and clinical aspects of cortisol as a biochemical marker of chronic stress. BMB Rep. 2015;48(4):209.\u003c/li\u003e\n\u003cli\u003eSteudte S, Kirschbaum C, Gao W, Alexander N, Sch\u0026ouml;nfeld S, Hoyer J, et al. Hair cortisol as a biomarker of traumatization in healthy individuals and posttraumatic stress disorder patients. Biol. Psychiatry. 2013;74(9):639-46.\u003c/li\u003e\n\u003cli\u003eLaufer S, Engel S, Lupien S, Knaevelsrud C, Schumacher S. The Cortisol Assessment List (CoAL) A tool to systematically document and evaluate cortisol assessment in blood, urine and saliva Compr. Psychoneuroendocrinol. 2022;9:100108.\u003c/li\u003e\n\u003cli\u003eRussell E, Kirschbaum C, Laudenslager ML, Stalder T, de Rijke Y, van Rossum EF, et al. Toward standardization of hair cortisol measurement: results of the first international interlaboratory round robin. Ther. Drug Monit. 2015;37(1):71-5.\u003c/li\u003e\n\u003cli\u003ePacella ML, Hruska B, Steudte-Schmiedgen S, George RL, Delahanty DL. The utility of hair cortisol concentrations in the prediction of PTSD symptoms following traumatic physical injury. Soc. Sci. Med. 2017;175:228-34.\u003c/li\u003e\n\u003cli\u003eAlghnam S, Alyabsi M, Aburas A, Alqahtani T, Bajowaiber M, Alghamdi A, et al. Predictors of seatbelt use among Saudi adults: results from the National Biobank Project. Front. Public Health. 2020;8:579071.\u003c/li\u003e\n\u003cli\u003eTaranov A, Puchkova A, Slominsky P, Tupitsyna T, Dementiyenko V, Dorokhov V. Associations between chronotype, road accidents and polymorphisms in genes linked with biological clock and dopaminergic system. Zhurnal nevrologii i psikhiatrii imeni SS Korsakova. 2017;117(4. Vyp. 2):28-33.\u003c/li\u003e\n\u003cli\u003eBlatt RJ. Banking biological collections: data warehousing, data mining, and data dilemmas in genomics and global health policy. Public Health Genomics. 2001;3(4):204-11.\u003c/li\u003e\n\u003cli\u003eLinsen L, Van Landuyt K, Ectors N. Automated sample storage in biobanking to enhance translational research: the bumpy road to implementation. Front. Med. 2020;6:309.\u003c/li\u003e\n\u003cli\u003eMalek A, Dadashzadeh H, Poorafkary N. Ranking of stressful life events in general population of Tabriz\u0026ndash;Iran. Med. J. Tabriz Univ. Med. Sci. Health Serv. 2009;30(4):73-80.\u003c/li\u003e\n\u003cli\u003eDadfar M, Kalibatseva Z, Lester D. Reliability and validity of the Farsi version of the Patient Health Questionnaire-9 (PHQ-9) with Iranian psychiatric outpatients. Trends Psychiatry Psychother. 2018;40(2):144-51.\u003c/li\u003e\n\u003cli\u003eWang X, Zuo Y, Jiang H, Yang L. Relationship between the incidence of road traffic accidents, psychological characteristics, and genotype in bus drivers in a Chinese population. Medical science monitor: Int J Exp Clin Res. 2018;24:5566.\u003c/li\u003e\n\u003cli\u003eGolestani M, Razzaghi A, Rezaei M, Vahedi L, Pourasghar F, Farahbakhsh M, et al. PERSIAN traffic safety and health cohort: a population-based precrash cohort study. BMJ Open. 2024;14(2):e080720.\u003c/li\u003e\n\u003cli\u003eSadeghi-Bazargani H, Shahedifar N, Somi MH, Poustchi H, Bazargan-Hejazi S, Jafarabadi MA, et al. PERSIAN Traffic Safety and Health Cohort: a study protocol on postcrash mental and physical health consequences. Inj Prev. 2022;28(3):269-79.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 623px;\"\u003e\n \u003cp\u003eTable 1. \u003cstrong\u003eResponsibilities of Biobank Key Members\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponsibilities\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eProgram Principal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003eCoordination with all committees, experts, and the software team; organizing intergroup meetings; financial management; overseeing the scientific and operational processes of the program; reporting; and article writing.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eExecutive Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003eProcuring necessary materials and equipment; duplicating and completing questionnaires; blood collection; preparing hair and blood samples; delivering samples to centers; serum preparation; biobank storage; sample control; coding; data entry into the software; reporting; and article writing.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eSoftware and Quality Control Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003eDeveloping software programs; quality control; assisting with data outputs; maintaining continuous communication between the software team and the executive secretary for accurate data entry and quality control; reporting; and article writing.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eSafety and Sample/Information Access Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003eSample storage; safeguarding patient information; adhering to ethical principles in sample preservation; communicating with other committees; and providing samples and data to researchers based on the ethical charter of the program.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eScientific Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 444px;\"\u003e\n \u003cp\u003eReporting; article writing; and defining and developing scientific projects.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\n \n\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 621px;\"\u003e\n \u003cp\u003eTable 2. Policies of Biobank Program\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eCost-effectiveness of the program\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eCollection of samples in a manner that allows a broad range of possible measurements\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eMinimization of time for collection, processing, transportation, and archiving of samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003ePrevention of potential sample degradation during transportation and archiving\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eQuality assurance (preventing errors and variability) and quality control methods (detecting errors and variability when they occur)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eEnsuring the security of samples and data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eManagement of sample and data usage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eManagement of sample and data sharing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eStandardization and centralization of processing at a dedicated site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 588px;\"\u003e\n \u003cp\u003eStaff training on agreed-upon and approved operational procedures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 33px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biobank, Hair, Cortisol, Stress, Road accident","lastPublishedDoi":"10.21203/rs.3.rs-8409982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8409982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChronic stress plays a contributing role in occurrence of traffic crashes and development of post-traumatic stress disorder (PTSD). Due to lack of a biobank for traffic injury victims in Iran, this study was conducted to develop a protocol for the design and establishment of a biobank for these victims.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis prospective cohort study will be conducted for a period of two years on individuals hospitalized due to traffic injuries at Imam Reza hospital in Tabriz University of medical Sciences/Iran. After extracting the details of individuals recorded in the Iranian Integrated Road Traffic Injury Registry SYSTEM (IIRTIRS), questionnaires on demographic characteristics, hair status, and life stressor events, and biological samples including hair and blood will be collected. Participants will be followed up after two month to complete the follow-up questionnaires. Qualitative and quantitative data (like hair cortisol levels) will be analyzed using the Chi-square test and ANOVA, respectively. Pearson correlation will be used to assess the association between hair cortisol levels measured by the enzyme-linked immunosorbent assay (ELISA) and chromatography methods.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe biobank of traffic crash victims in Northwest of Iran was designed and will be initiated by the Traffic and Health Hub, with collaboration from university-affiliated research centers and hospitals. As the first biobank of its kind in the country, its protocol has the potential to function as a blueprint for establishing similar biobanks in other developing countries.\u003c/p\u003e","manuscriptTitle":"Establishing a biobank for road traffic accident victims in Iran as a cohort study of stress related biomarkers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 08:28:42","doi":"10.21203/rs.3.rs-8409982/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68a3de52-7993-47c1-8a20-6d9c2caf1ae7","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T08:42:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 08:28:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8409982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8409982","identity":"rs-8409982","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}