Inclusion criteria

1. Cholestatic patients

   1. Age: ≤150 days, no gender limit.

   2. Diagnostic criteria for cholestatic jaundice: serum total bilirubin <85 μmol/L, but serum direct bilirubin ≥17 μmol/L or serum total bilirubin ≥85 μmol/L but direct bilirubin accounts for more than 20%.

   3. Gestational age ≥28 weeks, birth weight ≥1000 g.

   4. Sign the informed consent form.

2. Normal controls

   1. Age: ≤150 days old, no gender limit.

   2. Gestational age ≥28 weeks, birth weight ≥1000 g.

   3. No manifestations of cholestasis or other known hepatobiliary diseases.

   4. Sign the informed consent form.

Exclusion criteria

1. A history of severe hypoxia at birth, Apgar score <5 points.

2. A history of allergic diseases, inflammatory bowel disease and infectious diseases.

3. Complicated with other serious congenital malformations and immune deficiencies such as neurological, cardiovascular, pulmonary, endocrine and renal disorders that would interfere with the conduct and results of the study.

Sample collection

Blood samples of the cholestatic patients are obtained along with patients’ routine laboratory tests at the outpatient clinics or at the admission to the inpatient unit.

Blood samples of the normal controls are obtained along with patients’ routine laboratory tests at the admission to the inpatient unit from patients admitted for reasons other than hepatobiliary diseases.

The blood sample is centrifuged at 3000 rpm for 10 min to get the serum, and then the serum samples are stored at −80°C before measurement.

At the same time, a volume of 10 µL blood sample is obtained from the heel tip to a DBS patented by WuXi Diagnostics.15 After fully drying, it is stored at −20℃ before measurement.

Serum MMP-7 concentration measurement

Serum MMP-7 concentration is measured using an enzyme-linked immunosorbent assay (ELISA, R&D, DMP700 and WuXi Diagnostics’ Self-developed ELISA kit). Considering the measurement range of the kit, the serum samples of cholestatic patients are diluted 10 to 30 times. All the measurements are performed by WuXi Diagnostics who are blinded to other test results. Each sample is provided with three technical replicates in each assay, and the mean is recorded.

MMP-7 concentration measurement for DBS

Add 150 µl Calibrator Diluent (RD6-28) of the ELISA kit (R&D, DMP700) into the DBS tower, and then soak, shake (Thermo-shaker BE-9008) for 1 hour. Thereafter, 50 µl of the solvent extraction is loaded to detect the concentration of MMP7 on DBS using enzyme-linked immunosorbent assay (ELISA, R&D, DMP700). Each sample is provided with two technical replicates, and the mean is recorded.

Serum IL-33 concentration measurement

Serum IL-33 concentration is measured using enzyme-linked immunosorbent assay (ELISA, R&D, D3300B). Each sample is provided with three technical replicates, and the mean is recorded.

Serum bile acid profile measurement

A liquid chromatography-tandem mass spectrometry system (self-developed kit by WuXi Diagnostics) is used to detect the concentration of each bile acid in human serum including cholic acid (CA), glycocholic acid, taurocholic acid, chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid, tauroursodeoxycholic acid, lithocholic acid (LCA), glycolithocholic acid, taurolithocholic acid, taurohyocholic acid, hyodeoxycholic acid (HDCA), glycohyodeoxycholic acid, taurohyodeoxycholi acid, dehydrocholic acid.

Reference test

Intraoperative exploration and subsequent histological examination of liver biopsies are used to confirm the diagnosis of BA. If the gallbladders of some patients are seen to be completely atrophic, and the injection of contrast is hard to achieve, the baby will be diagnosed as BA at once. Otherwise, a cholangiography will be performed to further evaluate the anatomy of the intrahepatic bile ducts before a final diagnosis is made. Non-BA patients are confirmed by intraoperative cholangiography showing a patent biliary tree, percutaneous transhepatic biopsy excluding BA, genetic tests showing certain gene mutation or alleviation without surgical intervention during follow-up for at least 3 months. All the cases will be reviewed by the experts from the Chinese Biliary Atresia Collaborative Network before the final diagnosis is made.

Study flow

The study flow is depicted in figure 1. Infants who meet the eligibility criteria will be consecutively recruited. After informed consent has been taken, all participants undergo the index tests and the reference test. All the index tests are performed by investigators from WuXi Diagnostics who are blinded to other test results. The reference test is performed afterward by the investigators (experts in paediatric surgery or gastroenterology) of each hospital. Though the investigators of each hospital have access to the results of index tests before making the final diagnosis, the biases are limited since the reference standards are definite and objective. Following that, information is extracted from the medical record including the demographic parameters, routine liver function test, sonographic features, index tests, reference standard (diagnosis of BA or Non-BA) and outcome after treatment. A summary of the data variables collected in the DIABA-7 study is presented in table 1.

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Figure 1

Flowchart of the study patient. MMP-7, matrix metalloproteinase-7.

Data management

Data are collected at the time of measurement and from the medical record. Several steps have been taken to help ensure high-quality data collection. First, all DIABA-7 study investigators undergo standardised training before starting the study, including the sample collecting and storing, case report form filling. Besides, the electronic input is consistent with the content of the paper form. The data entry interface is established through Microsoft Access 2010 and uploaded to a central server on a weekly basis. Paper forms will also be available on the field as a backup.

Sample size

1. To clarify the normal reference range of serum MMP-7 and the diagnostic cut-off value in diagnosing BA, we set up the specificity of the test for distinguishing normal for case is 90%, using the formula: , where 0.9,

2. To establish the correlation between serum MMP-7 and DBS MMP-7, 150 cholestatic participants will be needed with a two-tailed α of 0.05 and β of 0.1, for achieving correlation coefficient r of 0.85.

3. To prove the efficiency of BA artificial intelligence (AI) multivariate diagnostic model is better than the single biomarker, 581 samples will be needed for each group, by comparing the AUC. Due to our group’s preliminary results and other literature, the hypothesis is:

H₀:

H₁:

where =0.956, =0.92.

Due to the statistical estimating above, the largest sample size request is 545 sample each group.

In conclusion, with the 5% data loss rate and the 5% screening fail rate, 1200 cases of cholestatic jaundice are proposed to be included, including 600 children with BA after intraoperative cholangiography (surgical gold standard) confirmation and 600 cholestatic children with other definite diagnosis than BA as well as 154 non-hepatobiliary disease controls to establish a reference interval.

Data analysis

Baseline characteristics of patients in the BA and non-BA groups are demonstrated using frequency distributions and descriptive statistics. Gender is described by n (%). Continuous variables are expressed as mean±SD, median and quartiles (Q1, Q3). In the univariate analysis, the χ² test will be conducted for gender, while Mann-Whitney U test will be performed for all continuous variables failing the Shapiro-Wilk W test for normality, otherwise, the student t test will be used. Multivariate logistic regression will be used to establish a diagnostic model based on several parameters.

Using the Lambda-Mu-Sigma (LMS) method, that is, using the median M (mu), the coefficient of variation S (sigma) and the Box-Cox conversion power L (lambda), which is required to convert the data to the normal distribution, the normal range of serum MMP-7 of each age group will be calculated.

Receiver operating characteristics (ROC) curves are constructed to calculate the best cut-off point and AUC for each index test. The sensitivity, specificity, PPV and negative predictive value are used to show the diagnostic accuracy.

Pearson correlation coefficient test will be applied to assess the correlation of serum MMP-7 and DBS MMP-7.

The least absolute shrinkage and selection operator regression method will be used to determine the optimal covariates to differentiate BA form non-BA. The patients will be divided into training set (70% data) and validating set (30% data) by random sampling function in R software. The covariates picked will be used to build the multivariable logistic regression model. The accuracy of the regression model will be assessed by the discrimination ability in the training set and the validating set.

Statistically significant difference was defined as a p value less than 0.05. All data analyses are performed using R software (3.6.3, Vienna Australia).

Patient and public involvement

Patients and their guardians are not involved in the study design and are not consulted to develop patient-relevant diagnoses and outcomes or interpret the results. Patients are not invited to contribute to the writing or editing of this document for readability or accuracy. We intend to disseminate the main results to the families of the trial participants and will pursue patient and public involvement in the development of an appropriate method to disseminate this information.

Ethics

This study was reviewed and approved by the Ethics Committee of Children’s Hospital of Fudan University (number 2020-296). The study will be performed in compliance with the Declaration of Helsinki and other relevant regulations.

The investigator has the responsibility to comprehensively introduce the purpose, procedures and possible risks of this study. An informed consent form must be given to each subject and be signed before recruitment.

The personal information will be kept confidential. Similarly, deidentified data are used for statistical evaluation. Only the investigators can identify the subject’s name and other personal information by initials.

Disseminations

Dissemination will be guided by investigators and patients. The aim is to publish the study results in a high-quality peer-reviewed journal and present the findings at international academic meetings.