Full-thickness versus rectal suction biopsy for the diagnosis of Hirschsprung’s disease: study protocol for a prospective, randomized open-label, within-patient crossover trail

Abstract

Background

Hirschsprung disease (HD) is a congenital malformation characterized by the absence of ganglion cells in the bowel wall. The aganglionosis extends from the rectum and proximally into the gastrointestinal tract in various lengths. The diagnosis relies on histological examination of rectal biopsies. Two main biopsy techniques: rectal suction biopsy (RSB) or full-thickness biopsy (FTB) are available. They differ in the degree of invasiveness, postoperative complications and diagnostic accuracy.

Methods

This is a prospective trial including 300 patients aged 0–17 years scheduled for rectal biopsy on the suspicion of HD. Each patient will undergo both RSB and FTB and thus serve as its own control. The sequence of histological analysis of RSB and FTB will be randomized. The primary objective is to compare the diagnostic performance of FTB and RSB in children evaluated for Hirschsprung’s disease. Specifically, the aim is to assess how effectively each technique obtains adequate tissue samples for nerve cell evaluation and to compare the rates of inconclusive biopsies associated with each method. The secondary objective is to evaluate the concordance to the National Institute for Health and Care Excellence (NICE) symptom-based criteria for rectal biopsy in children with severe constipation.

Discussion

The findings from the BIOPSY-STUDY are expected to provide high-quality evidence to guide biopsy selection in suspected Hirschsprung’s disease. By directly comparing RSB and FTB within the same patients, the study aims to clarify diagnostic accuracy, reduce inconclusive results, and support more efficient, cost-effective clinical pathways. Despite being single-center, the rigorous design enhances internal validity and clinical relevance.

Trial registration

The study is registered at ClinicalTrials.gov NCT05307419. Registered on April 01, 2022.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13063-026-09817-x.

Administrative information

Title {1}	Full-thickness versus rectal suction biopsy for the diagnosis of Hirschsprung’s disease: protocol for an open-label-crossover study
Trial registration {2a and 2b}	ClinicalTrials.gov (ID: NCT05307419)
Protocol version {3}	July 2025, version 1
Funding {4}	Additional procedure-related costs and Ph.D. stipend for primary investigator (NB) will be applied for from public as well as private funds. The study is not economically supported by the pharmaceutical industry. Grants will be handled by Odense University Hospital. The research account will be subjected to public audit. There will be no remuneration to participant None of the contributors to this protocol have financial or other competing interests to declare
Author details {5}	Niels Bjørn, MD, Ph.D.-student, consultant pediatric surgeon (NB) Gunvor Madsen, MD, consultant pathologist GM) Rasmus G. Nielsen MD, Ph.D., consultant pediatrician (RGN) Andreas Kristian Pedersen (AKP) Niels Qvist, MD, DMSci, Professor (NQ) Mark Bremholm Ellebæk, MD, Ph.D., Professor, consultant pediatric surgeon (MBE)
Name and contact information for the trial sponsor {5b}	Mark Bremholm Ellebæk Odense University Hospital, J.B. Winsløws Vej 4, 5000, Odense C, Denmark E-mail: mark.ellebaek1@rsyd.dk
Role of sponsor {5c}	The sponsor has the final authority over study design and overall responsibility in relation to legislation, administration, funding, and implementation of the project

Introduction

Background and rationale {6a}

Hirschsprung disease (HD) affects approximately 1 in 5000 newborns and is characterized by the absence of ganglion cells in the bowel wall. The aganglionosis extends from the rectum and proximally into the gastrointestinal tract in various extensions. Clinically the condition is characterized by delayed meconium passage (more than 24 h), defecatory straining, constipation and fecal impaction above the aganglionic segment [1]. Diagnosis relies on histological demonstrated aganglionosis in a rectal biopsy. The demonstration of nerve trunk hypertrophy strengthens the diagnosis [2, 3].

Two main biopsy techniques are commonly employed: full-thickness rectal biopsy (FTB) or rectal suction biopsy (RSB). RSB has become the most common method because it is less invasive and may be performed without the need for general anesthesia as opposed to FTB [4]. The complication rate seems to be similar [5] but inconclusive biopsy rates for RSB range from 10 to 40%, whereas FTB has less than 6% inconclusive biopsies [6–8].

The National Institute for Health and Care Excellence (NICE) recommends rectal biopsy in infants and children presenting with specific clinical features suggestive of Hirschsprung’s disease, including delayed passage of meconium (more than 24 h after birth), chronic constipation since infancy, abdominal distension, vomiting, and failure to thrive. Verification of these criteria is essential to support consistent diagnostic decision-making, ensure high-quality care, and to optimize patient outcomes [9].

Beyond diagnostic accuracy, the cost of RSB versus FTB is another important question that to our knowledge has not been studied. The higher incidence of inconclusive biopsies with RSB will result in re-do procedures, which together with a more tedious histological examination may increase overall costs.

Objectives {7}

The objective of this study is to compare two biopsy methods, RSB and FTB, in terms of their ability to provide diagnostically adequate tissue samples to evaluate the presence or absence of ganglion cells and to assess the rate of inconclusive procedures associated with each technique. We have designed this as a Superiority study on the hypothesis that full-thickness biopsy is superior to RSB.

The secondary objectives are to:

1. Compare the size and quality of each biopsy, in terms of the different layers of the rectal wall included.

2. Assess if the clinical indication for biopsy is in alignment with the National Institute for Health and Care Excellence (NICE) [9] symptom-based criteria.

3. Compare the direct procedural costs associated with FTB and RSB, respectively.

Trial design {8}

This study is a prospective randomized open-label, within-patient-crossover trial with a superiority framework designed to compare the rate of inconclusive biopsies between RSB and FTB in diagnosing Hirschsprung’s disease.

We have designed this as a superiority study on the hypothesis that full-thickness biopsy is superior to RSB.

Each patient will undergo both biopsy procedures under the same general anesthesia and the sequence for the primary histological examination (RSB or FTB) will be randomized.

If the initial biopsy is inconclusive, the other biopsies will be evaluated immediately for diagnostic purposes.

If the initial result is conclusive, the second sample will be stored and assessed after 30 days to evaluate diagnostic concordance. The pathologist will be blinded for the result of the primary investigation.

Methods: participants, interventions, and outcomes

Study setting {9}

The study will be anchored in the Research Unit for surgery at the Department of Surgery Odense University Hospital (OUH). The study will be conducted at Pediatric Surgical Department, Odense University Hospital, with coordinated participation from the Departments of Pediatrics and Pathology.

All departments have agreed to participate in this project. All the clinically related study procedures are well established at the hospital.

Eligibility criteria {10}

Patients referred for rectal biopsy due to suspected Hirschsprung’s disease (HD) will be considered eligible if they meet the inclusion and exclusion criteria. Inclusion criteria are patients aged between 0 and 17 years who are referred for biopsy on suspicion of HD, and where informed consent has been obtained from parents or legal guardians. Patients will be excluded if they have undergone previous anorectal surgery.

Who will take informed consent? {26a}

Parents or guardians will receive both written and oral information on the project, the rectal biopsy procedures, and their right as a project participant. Information will be provided by the pediatric surgeon in the outpatient clinic. Informed consent must be obtained before any surgical scheduling. For hospitalized children requiring subacute biopsy, written and oral information will be provided upon admission with formal consent secured prior to scheduling the procedure.

Additional consent provisions for collection and use of participant data and biological specimens {26b}

By the inclusion, the parents or guardians will be informed that by giving informed consent the responsible medical doctor or a person specially designated and instructed to conduct the work on behalf of the responsible medical doctor, has access to and may collect information about the patient’s health including history of disease symptoms and other health issues, present physical and medical status and the results of the medical and histological examinations.

Preoperatively, access to the patient’s medical journal will allow confirming the inclusion/exclusion criteria. When the final histological diagnosis is established the information from the patient record will be used to evaluate whether the indication for the biopsy complies with the National Institute for Health and Care Excellence (NICE)’s symptom-based criteria for rectal biopsy in children with severe constipation [9].

Tissue specimens collected for this trial will be stored in the pathology department’s tissue bank according to standard procedures. The parents or guardians will by consent allow future use in connection with the project or other relevant projects in compliance with approval from the Sponsor and relevant ethical approvals.

Anonymized participant data may be shared with other researchers upon written approval from the Sponsor and in compliance with relevant ethical approvals. Any use of the data must acknowledge the data source and funding body. Anonymized datasets may also be deposited in public repositories, ensuring that participants cannot be identified.

Interventions

Explanation for the choice of comparators {6b}

The comparators, full-thickness biopsies and rectal suction biopsies have been chosen, as they are the standard biopsy method currently employed in the diagnosis of HD [6–8].

Punch biopsies are an alternative technique that has demonstrated results comparable to those of rectal suction biopsy. However, not yet adopted as widely as RSB and FTB [10].

Intervention description {11a}

All procedures will be performed under general anesthesia, with both the RSB and FTB conducted during the same intervention. RSB will precede FTB to avoid overlapping sampling sites. All patients will be followed postoperatively for complications based on the Clavien-Madadi classification [11], with a 30-day follow-up.

Surgical procedure

Rectal suction biopsy

Rbi2 Suction Rectal Biopsy System (Aus Systems™, Australia) is inserted through the anus, and biopsies are taken at different levels above the dentate line, first biopsy at 2, 4, and 6 cm above the dentate line in the posterior aspects of the rectum.

Full-thickness biopsy

With a speculum inserted to the anus a suture including the rectal wall is placed in the posterior aspects of the rectum, approx. 2 cm above the dentate line. The rectal wall is raised by traction on the suture and an approximately 5 mm large FTB is cut with a pair of scissors. Hemostasis is secured by bipolar electrocoagulation, and the defect is sutured with absorbable suture (Vicryl® 3-0).

Postoperative follow-up

All parents/guardians will be informed about biopsy results as part of standard care within 2–3 weeks. Results will be communicated either by letter or phone, depending on the parents’ preferred method of contact. A chart review will be conducted 30 days postoperatively to assess and document complications, classified according to the Clavien–Madadi [11] system. This review will support risk assessment and interim analysis in accordance with the requirements of the ethical review board.

Criteria for discontinuing or modifying allocated interventions for a given trial participant {11b}

If any unexpected or serious adverse events occur during the study, the trial will be immediately paused and reviewed by the responsible medical investigators and the leadership of the Research Unit for Surgery. If necessary, the relevant ethics committee will be notified in accordance with applicable regulatory requirements.

Serious adverse events will be monitored and assessed through interim analyses, particularly in relation to the biopsy procedure and associated complications. Should any new findings emerge, that may influence the diagnostic pathway, appropriate amendments will be made to the study protocol.

Participation in the study is entirely voluntary. Declining participation or withdrawing consent at any stage will not affect the patient’s access to standard care or treatment. Parents or guardians may withdraw their consent at any time without consequences for the child’s ongoing medical care.

Strategies to improve adherence to the intervention {11c}

To ensure high adherence to the study protocol and minimize participant dropout, several strategies will be implemented. Clear, parent-focused information will be provided to families before enrollment, including detailed explanations of the biopsy procedures, potential risks, and the importance of completing all planned evaluations. Ongoing communication between clinical staff and families will be prioritized to maintain engagement and address concerns proactively.

Scheduling flexibility and coordination with standard clinical visits will be used to reduce participant burden. Any discomfort or concerns related to the biopsy procedures will be managed promptly. In case of adverse events or protocol-related uncertainty, participants and families will receive direct updates and support throughout the decision-making process.

These measures are designed to support voluntary and informed participation, reduce anxiety, and encourage continued involvement, ultimately contributing to the reliability and completeness of study data.

Relevant concomitant care permitted or prohibited during the trial {11d}

A single dose of perioperative antibiotics will be administered peroperatively, in accordance with departmental guidelines. Postoperative medical management will be carried out by the attending pediatrician typically involving continuation of preoperative medications or extension of prophylactic antibiotic therapy if clinically indicated. As part of standard care, no rectal medications or instrumentation will be permitted during the first three postoperative days.

Provision for post-trial care{30}

Participants who experience harm or injury as a direct result of study procedures will be covered under the Danish healthcare system’s existing compensation framework. In accordance with national regulations, patients are eligible to seek compensation through Patienterstatningen (the Danish Patient Compensation Association). This system ensures that participants can apply for compensation without having to prove fault, provided the injury meets the criteria set out in Danish law.

No additional insurance coverage is required beyond this statutory scheme, and all participants will receive standard medical care throughout and after the trial, regardless of participation status or study outcomes.

Outcomes {12}

Primary outcome

Proportion of conclusive histological diagnoses obtained from FTB and RSB in children undergoing evaluation for suspected Hirschsprung’s disease.

Secondary outcomes

Tissue quality and size: Comparison of the size and the presence of the relevant layers of the rectal bowel wall in tissue samples obtained by FTB and RSB.

Clinical indication alignment: Proportion of included patients fulfilling indication for biopsy in alignment with the National Institute for Health and Care Excellence (NICE) criteria for the diagnosis and management of children with constipation.

Cost analysis: Comparison of the pathological procedural costs associated with FTB or RSB as the primary diagnostic procedure.

Participant timeline {13}

Participant timeline is specified in Fig. 1 below.

Fig. 1.

Participant timeline

Sample size {14}

Sample size calculations are based on data from the largest published series [5, 6], which report an inconclusive biopsy rate of 5% for FTB and 10% for RSB. Using McNemar’s test, a sample size of 300 patients is required to detect this difference with 70% power, a 5% significance level, and an anticipated dropout rate of 5%.

Recruitment {15}

Parents or guardians of children referred to rectal biopsies on the suspicion of HD, and fulfilling the inclusion criteria will be approached by the study responsible and informed about the purpose and design of the study. This will be done in the hospital outpatient clinic or as a telephone call/video consultation. The information will be given in a calm environment and will be given both orally and written (see Appendix 1—Patient information). The parents/guardians will have the possibility to involve relatives or other lay people in the information and discussion of the project. They will be given the possibility to further discuss the project with the clinical responsible researcher either by phone or face-to-face at the outpatient clinic and have at least 24 h to consider participation. The informed consent must be documented in the patient’s electronic medical record prior to any planning of surgical biopsy procedure.

Assignment of interventions: allocation

Sequence generation {16a}

All patients will undergo RSB followed by FTB under general anesthesia. Afterwards, the tissue evaluation will be randomly assigned to either Group A: primary evaluation of RSB samples, or Group B: primary evaluation of FTB.

Concealment mechanism {16b}

The randomization process will be Web-based using the RedCap™ Randomization Module hosted by Open Patient data Explorative Network (OPEN). Immediately after the biopsy is taken, randomization will be carried out using the REDCap™ Randomization Module to determine whether the RSB or the FTB will be assessed first by the pathologist. This ensures that the randomization process does not influence the biopsy technique and reduces potential procedural bias.

Randomization will be 1:1. The system is managed by the primary investigator, and the assigned sequence, whether RSB or FTB must be evaluated first.

Implementation {16c}

The study will be integrated into routine clinical workflows at the participating center. Eligible participants will be identified during their referral process. After informed consent is obtained, the lead investigator will enroll the participant into the study.

The study team will be trained to incorporate these steps seamlessly into daily clinical practice, ensuring that participation does not delay or interfere with standard diagnostic or treatment timelines.

Assignment of interventions: blinding

Who will be blinded {17a}

This case-controlled trial incorporates blinding at multiple levels to reduce bias: Parents/guardians will be blinded to the randomization sequence, i.e., the order in which the RSB and FTB are evaluated by the pathologist. Pathologists performing the control histological re-evaluation at the 30-day follow-up will be blinded to the initial pathology findings and the clinical diagnosis assigned after the first assessment. The trial statistician will be blinded to the randomization sequence, ensuring objective statistical analysis of outcomes without knowledge of which biopsy was evaluated first. These blinding procedures are implemented to minimize confirmation and observer bias, and to ensure that clinical decisions, including postoperative management and follow-up, are not influenced by the initial order of pathological evaluation.

Procedure for unblinding if needed {17b}

In cases where unblinding becomes necessary for reasons such as patient safety or other concerns, the primary investigator will make this decision if needed.

Data collection and management

Plans for assessment and collection of outcomes {18a}

Data will be collected using electronic case report forms in REDCap™ hosted by Odense Patient data Explorative Network (OPEN). Data will be collected using electronic case report forms (eCRFs) in REDCap™, hosted by the Odense Patient data Explorative Network (OPEN). The study lead will be responsible for overseeing data collection and ensuring data quality. Systematic data validation rules will be applied within REDCap. Additionally, periodic data audits will be performed to identify inconsistencies or missing values. All personnel involved in data entry will be trained in standardized procedures to further safeguard data integrity.

Plans to promote participant retention and complete follow-up {18b}

To support retention and minimize loss to follow-up, study visits will align with routine clinical appointments. If a participant discontinues or deviates from the intervention, key outcome data will still be collected where possible, unless consent is withdrawn. All discontinuations will be documented to maintain data integrity.

Data management {19}

All data is stored in OPEN in accordance with current GDPR guidelines. The project is subject to a data processor agreement (Data Processing Agreement, DPA; Approval number 20/7078).

To ensure data accuracy and integrity, a double data entry procedure (double control) will be implemented. This involves two independent entries of the same data by separate individuals, followed by an automated comparison to identify and resolve discrepancies. This quality control step minimizes data entry errors and enhances the reliability of the dataset for analysis.

Confidentiality {27}

The study complies with all applicable General Data Protection Regulation (GDPR) requirements. Each participant is assigned a unique study identification number at the time of screening to ensure data anonymization. Access to identifiable information is restricted to authorized personnel and is disclosed to third parties only with participant consent or when required by law. Trial results will be reported in aggregate, without any information that could identify individual participants.

Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}

Histological handling of biopsies

Staining procedures

For histological evaluation of rectal biopsies, all tissue samples are first fixed in 10% neutral buffered formalin, then dehydrated, cleared, and embedded in paraffin. Thin Sects. (3–5 µm) are cut, mounted on slides, and stained with hematoxylin and eosin (H&E).

The immunohistochemistry staining is performed according to the following protocol: 4-µm sections are cut and mounted on FLEX IHC Microscope slides (Dako/Agilent, Glostrup, Denmark). Sections are dried at room temperature and baked at 60 °C for 60 min before immunohistochemical staining. Staining is automated at the BenchMark Ultra immunostainer (Ventana Medical Systems, Tucson, AZ) using the OptiView-DAB detection (Ventana Medical Systems, Tucson, AZ). Primary antibodies, dilutions, incubation times and epitope retrieval procedures. Nuclear counter staining is performed using Hematoxylin II (Ventana Medical Systems, Tucson, AZ) at the BenchMark Ultra instrument. Finally, slides are washed, dehydrated, and cover slipped using an automated Dako coverslipper (Dako/Agilent, Glostrup, Denmark).

Full-thickness biopsies

Tissue specimens from FTB will be evaluated for size. The biopsies will be embedded in paraffin and sectioned into 5 µm slices for histological evaluation. The initial preparation includes one slide with three serial sections stained with hematoxylin and eosin (HE), one slide stained for S-100 and one for calretinin. The presence of relevant tissue layers, including mucosa, submucosa, muscle, and serosa will be registered.

If ganglion cells (GCs) are identified on initial microscopy of the HE stained section, the diagnosis is considered negative for Hirschsprung’s disease (HD), and the biopsy is classified as conclusive.

If ganglion cells (GCs) are not identified in the initial biopsy, or the sample is deemed inconclusive, further evaluation is then performed on the preserved sample, regardless of whether it is from a RSB or a FTB, depending on which was investigated first. Staining with S-100 and calretinin is used to support the diagnosis. S-100 highlights hypertrophic nerve trunks, which may indicate aganglionosis. Calretinin staining is positive whenever ganglion cells are present independent of the presence of nerve hypertrophy.

Rectal suction biopsy

For the RSB, the three biopsies obtained will be evaluated for both size (largest diameter in mm) and the presence of relevant tissue layers, including mucosa, submucosa, muscle, and serosa. Two slides per biopsy are prepared, each containing 5–8 serial HE-stained sections, resulting in a total of 10–16 sections per biopsy. A biopsy sample is considered suitable for further diagnostic assessment only if it contains an adequate amount of submucosa. The highest-quality sample meeting this criterion is selected for immunohistochemical staining with S-100 and calretinin. The presence of ganglion cells confirms a conclusive diagnosis.

Statistical methods

Statistical methods for primary and secondary outcomes {20a}

Software

Analyses will be performed using Stata version 18 or later and R version 4.4 or later, depending on the statistical technique required.

Primary outcome analysis (conclusive diagnosis rates)

McNemar’s test will be used to compare the proportion of conclusive outcomes between RSB and FTB, as the primary outcome is binary (conclusive vs. inconclusive) and based on paired data from each participant. In addition to the hypothesis test, exact 95% confidence intervals will be reported for the proportion of conclusive results for each biopsy method. This approach allows both a comparison of diagnostic yield and an estimation of precision around each method’s conclusiveness rate.

Logistic mixed effects regression

Gives the probability of obtaining a conclusive result as a function of biopsy type (RSB vs. FTB) while adjusting for patient-specific covariates (e.g., age or any other factors affecting biopsy quality). We will in the model take into account the clustering by patient as a random intercept in the linear part of the logistic regression. The association between method and outcome across age groups will be assessed using logistic regression.

Mixed effects linear regression or median regression

For continuous measures of sample quality depending on the normality of the data distribution. A random intercept for each patient will be included.

Interim analyses {21b}

Interim analyses will be conducted after 25%, 50%, and 75% of participant enrollment. These will assess the primary outcome as well as adverse event rates. If the complication rate exceeds 8%, the study will be reviewed by the internal monitoring committee and, if appropriate, the independent safety committee. To control for type I error inflation due to multiple analyses, Group Sequential Methods (e.g., O’Brien-Fleming boundaries) will be applied.

Methods for additional analyses (e.g., subgroup analyses) {20b}

Prespecified subgroup analyses will assess whether the diagnostic yield and cost-effectiveness of RSB compared with FTB vary according to patient age (< 6 months, 6–24 months, > 24 months), tissue quality and number of histological layers, fulfillment and number of NICE diagnostic criteria, occurrence of complications, and the need for rectal irrigation. Conclusive diagnosis rates between RSB and FTB will be compared using McNemar’s test, and potential effect modification will be evaluated by including interaction terms between biopsy type and subgroup variables in logistic mixed-effects regression models. For continuous outcomes such as tissue quality metrics, mixed-effects linear or median regression will be applied with similar interaction terms. Given the limited statistical power and the potential for type I error due to multiple comparisons, these analyses will be considered exploratory and hypothesis-generating. Statistical significance will be defined as a two-sided p-value < 0.05, with all analyses performed in Stata. A detailed analysis plan, including methods to address potential confounding, will be finalized prior to data processing. A detailed statistical analysis plan will be finalized before data processing, including strategies to account for potential confounding variables.

Cost-effectiveness analysis (CEA): Compare the costs per conclusive diagnosis between RSB and FTB, potentially plotting cost-effectiveness acceptability curves. This analysis will be performed by mixed-effects regression models taking multiple samples from the same patient into account. Moreover, due to the fact that cost data are often skewed, bootstrapping with 1000 repetitions will be performed to provide confidence intervals around the cost-effectiveness ratios to account for distributional challenges.

Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}

A per-protocol analysis will be undertaken that excludes participants who did not adhere to the protocol. An intention to treat analysis will be done on participants with some missing data. Missing observations from patients will be handled by the inherent weighting of measurements included in mixed-effects regression, assuming missing at random conditional on included covariates.

Plans to give access to the full protocol, participant level-data and statistical code {31c}

The full protocol, de-identified participant-level data, and the data analysis will be made available upon reasonable request, following publication of the primary results.

Oversight and monitoring

Composition of the coordinating center and trial steering committee {5d}

The coordinating center is the Research Unit for surgery at the Department of Surgery Odense University Hospital (OUH). The Trial steering committee will be comprised of the primary investigator, sponsor and local center leads. Monthly or bi-monthly meetings will ensure smooth communication and workflow.

Composition of the data monitoring committee, its role and reporting structure {21a}

The data monitoring committee will consist of lead investigator, sponsor and a statistician connected to The Research Unit for surgery as well as a data manager from Odense Patient data Explorative Network with extensive expertise in data management. Meetings will be held if necessary but close communication will be upheld throughout the study.

Data monitoring {21b}

A Data and Safety Monitoring Committee (DSMC) consisting of statisticians, data managers, and study investigators will oversee data quality and protocol compliance. The independent safety committee, with representation from the GAIN Advisory Board [12], will meet on an as-needed basis in response to safety signals or major protocol deviations. This committee has the authority to recommend continuation, modification, or termination of the trial.

The safety committee, consisting of members from the GAIN Advisory Board will only meet when necessary [12].

Adverse event reporting and harms {22}

Reported adverse and serious adverse events, defined as greater than grade IIIA postoperative complications according to the Clavien–Dindo classification, will be discussed within the safety committee and appropriate action will be taken. Normally, the distinction between serious and non-serious adverse events follows the definitions provided in the GCP-guidelines, which align with standard criteria used in pharmacovigilance. Serious adverse events typically include outcomes such as death, life-threatening conditions, unplanned hospitalization or its prolongation, significant disability, or congenital anomalies. All adverse events will be monitored and documented in accordance with these definitions.

Frequency and plans for auditing trial conduct {23}

There are no plans for independent auditing of trial conduct beyond routine monitoring by the study team. Interim safety analyses will be performed when 25%, 50%, and 75% of participants have been enrolled, focusing on adverse events classified according to the Clavien-Madadi (11) classification or other safety concerns arising during the study period.

Plans for communicating important protocol amendments to relevant parties (e.g., trial participants, ethical committees) {25}

All significant amendments to the research protocol will be promptly communicated by the steering committee to the appropriate stakeholders. This includes the Scientific Ethical Committee, the Chief of the Surgical Department, the Safety Committee, and all other relevant institutional and regulatory parties.

Before implementation, any protocol modifications will undergo formal review and approval by the appropriate ethics committee. Following ethical approval, the amendments will also be submitted to site-specific institutional authorities and regulatory bodies, as required. Clear documentation and version control of all protocol changes will be maintained to ensure transparency, traceability, and regulatory compliance throughout the study.

Dissemination plans {31a}

The principal investigators are committed to communicating the results, negative, positive, or inconclusive, at local, national and international scientific meetings. The findings will be published in well-established, international peer-reviewed open access journals. After the end of the study, data will be uploaded to an open repository.

Discussion

The BIOPSY-STUDY addresses critical diagnostic challenges in the evaluation of RSB and FTB for suspected Hirschsprung’s disease (HD). It aims to identify the most efficient method for reducing inconclusive biopsy rates, improving diagnostic accuracy, and minimizing healthcare resource use. The results have direct implications for patient care, potentially reducing the need for repeat procedures, expediting diagnosis, and lowering diagnostic costs in this vulnerable patient population.

While RSB is less invasive and widely utilized, it has been associated with higher inconclusive rates compared to FTB, leading to delayed diagnosis and additional healthcare burden [5, 6]. Most previous studies have been retrospective. To our best knowledge this study will be the first prospective randomized trial, using a paired, self-controlled design where each patient undergoes both biopsy types. This design is expected to provide the highest level of evidence, minimizing inter-patient variability and strengthening internal validity.

Several clinical scenarios may arise based on the study findings. If the two methods are shown to be equivalent, clinicians could prioritize the less invasive RSB, optimizing patient comfort and procedural efficiency. If one method demonstrates superior diagnostic reliability, clinical pathways would need to adapt accordingly to incorporate the more accurate approach, ensuring that children with suspected HD receive the most definitive and timely diagnosis possible. In either case, the findings will offer robust, evidence-based guidance for standardizing biopsy choice in clinical practice.

Evaluating alignment with NICE guidelines is of importance, as relevant and standardized diagnostic practices are of clinical importance to ensure high quality in selection of patients for biopsy. By assessing adherence to established, evidence-based guidelines, the study aims to support more accurate and consistent clinical decision-making and improve overall patient care [9].

Economic aspects remain a significant consideration. Previous studies suggest that while RSB may have lower upfront procedural costs, inconclusive results can lead to greater cumulative expenses due to repeated investigations [4]. The BIOPSY-STUDY will systematically assess the pathological costs associated with each method, providing important data for healthcare decision-makers regarding cost-effectiveness and resource allocation.

The study has several limitations. First, the trial is conducted at a single center, with only two pathologists evaluating the specimens, which may limit external generalizability. Although having few evaluators enhances internal consistency, it may not fully represent broader variability in pathological interpretation across different institutions. Additionally, the introduction of a new biopsy technique (RSB) at our center, during the study period may impact the results. However, thorough documentation and stratification based on the specific procedural technique used will help mitigate this potential source of variability.

As with all randomized controlled trials (RCTs), the trial may face challenges such as patient selection bias, loss to follow-up, or unmeasured confounding factors despite randomization efforts.

In conclusion, the BIOPSY-STUDY will be the first randomized, self-controlled prospective study directly comparing RSB and FTB for the outcome of inconclusive biopsy result, when evaluating for HD diagnosis. Its design provides the strongest available evidence to inform clinical decision-making. The study findings have the potential to directly impact patient management by guiding biopsy selection, optimizing diagnostic accuracy, minimizing delays, and improving cost-effectiveness in the diagnostic pathway for children suspected of having HD.

Trial status

Recruitment began on October 1, 2022, and the trial will continue for an estimated four to five years. There are included 200 patients (21-04-2026). No adverse events have been observed. 25% and 50% interim analyses in process.

Authors’ contributions {31b}

NB is the lead investigator; he conceived the study, led the proposal and protocol development. MB will act as sponsor and has contributed greatly to proposal and protocol development. RGN, GM, and NQ contributed to study design and to development of the proposal. All authors read and approved the final manuscript.

Funding {4}

Open access funding provided by University of Southern Denmark Any private companies will not financially or in any other ways support the research project.

Region of Southern Denmark, University of Southern Denmark and Odense University Hospital are covering financial support as Ph.D. stipendium costs for Niels Bjørn.

The patients will not receive economic compensation for their participation.

Received funding will be transferred to the department’s research account in Danske Bank,

Account no.: 4597 11463924.

Odense University Hospitals CVR no.: 29766797.

All funding will be administered by “forskerservice” at SDU. And the account will be under public revision?

Data availability {29}

During the study, all data will be securely stored in an OPEN-REDCap database hosted by Odense Patient data Explorative Network (OPEN), in compliance with GDPR regulations and institutional data governance policies. Upon study completion, an anonymized version of the final dataset will be prepared and an application will be submitted for its long-term storage in a publicly accessible research data repository at Rigsarkivet (The Danish National Archives). This will allow for future research use, subject to appropriate data access controls and ethical oversight.

Declarations

Ethics approval and consent to participate {24}

Ethical approval has been granted by the Region of Southern Denmark Research Ethics Committee S-20190116. Study findings will be published in peer-reviewed scientific journals and presented at relevant conferences. Anonymized data will be made available upon reasonable request.

Previous studies have shown that performing two full-thickness biopsies during a single procedure is safe in children [7]. Therefore, we do not anticipate any increased risk associated with the addition of up to three suction biopsies during the same intervention.

Consent for publication {32}

Consent for publication will be obtained from all participants prior to inclusion in the study. See Appendix 1 and 2 as stated in the consent forms. No personal data will be published.

Competing interests {28}

The authors declare that they have no competing interests.