digital supplemental content 1

(1)

DIGITAL SUPPLEMENTAL CONTENT 1 Operational Research Definitions

Operational research definitions are meant for retrospective identification of clinically evident (CE) manifestations and/or events, with the understanding that pathophysiologic development of these manifestations/events may occur prior to meeting criteria of the research definition.

For example, increases in portal pressure (e.g. portal hypertension) occur before it is clinically evident. Some definitions have >1 level of rigor (e.g. definite vs possible).

Chronic Liver Disease Manifestations Failure-to-thrive (FTT)

Research Definition:

- Clinically evident: Weight-for-age Z-score of < -2.0 using World Health Organization (WHO) growth standards, captured at least 3 months post-HPE

Despite being a common underlying indication for LT in patients with BA (1-7), there is currently no definitive criteria for diagnosis of failure-to-thrive (FTT) in children with chronic liver disease (CLD). For this study, FTT in BA was defined as a documented weight-for-age Z-score <-2.0 based on previously published WHO pediatric standards (8). Associated nutritional data

collected included caloric density of formula, medium-chain triglycerides supplementation, and use of parenteral-nutrition, either exclusive or non-exclusive, for a duration of over 1-month. In order to avoid the immediate post-surgical period, all Z-scores and nutritional information was collected at a cutoff of at least 3 months after the date of HPE. Using pre-set age-adjusted growth charts (available through the electronic medical record system), Z-scores were adjusted appropriately based on gestational age.

Traditionally, a weight-for-age Z-score below -2.0 have been designated as “wasting” by the WHO, and previous reports have associated Z-scores near or below -2.0 with poorer native liver survival in children with CLD (2, 3, 6, 9). Though it is acknowledged that weight in BA may be confounded by manifestations such as ascites or organomegaly (5, 7), review of existing literature and expert opinion review suggests that a cutoff of -2.0 is likely sufficient for identification of obvious, clinically evident FTT. Separate methods have been developed to identify ascites (see definition below), so that it would not confound the assignment of FTT as a CLD manifestation. It is not known how organomegaly may impact on this parameter.

Anthropometric measurement such as triceps skin folds and mid-arm circumference (5) were not included in our research definition, as neither was performed frequently enough in the patient cohort for accurate longitudinal assessment of nutritional status.

(2)

Portal Hypertension Research Definition (10):

- Definite clinically evident portal hypertension (dCEPH):

1. Documented CLD-manifestation of either clinically evident ascites (clinically diagnosed ascites necessitating chronic diuretic therapy) or varices(per endoscopic evidence)

OR

2. Documented combination of clinical findings of splenomegaly¹AND thrombocytopenia²

1. Documented >2 cm below costal margin on physical exam OR enlarged per imaging report based on normal standards for age)

2. Platelet count <150000/ml for at least two consecutive measurements, without evidence of immune thrombocytopenic purpura

- Possible clinically evident portal hypertension (pCEPH):

1. Documented clinical finding of only splenomegaly in the absence of a CLD- manifestation (e.g. varices or CE ascites)

2. Documented clinical finding of only thrombocytopenia in the absence of a CLD- manifestation (e.g. varices or CE ascites)

Portal hypertension (PH) remains a significant CLD manifestation in the disease course of BA (10-12), despite earlier identification of disease (13, 14) and successful intervention by HPE (15). For this study, we implemented the previously published research definition clinically evident portal hypertension (CEPH) (10). While clinically-significant PH in adults can be identified and defined by a hepatic venous pressure gradient of >10 mmHg, the direct or indirect measurement of portal pressures is rarely done in children. This pediatric research definition, which incorporates the identification of complications (ascites, varices) or clinical findings (thrombocytopenia and splenomegaly), is easily reproducible in both retrospective and prospective studies (10) , with an earlier version previously able to identify CEPH in 2/3 of a moderately sized pediatric BA cohort (12).

Ascites

- Definite clinically-evident ascites (dCE ascites): Inappropriate weight gain¹ with subsequent appropriate response by weight loss² to diuretic administration³ in the setting of at least one clinical finding (physical exam or ultrasonographic evidence) attributable to the clinical diagnosis of ascites⁴

1. Inappropriate weight gain velocity per WHO standards (g/day) ³95^th percentile between two weight measurements, at least 24 hours apart

2. Weight loss: a reduction of weight by at least 50% of inappropriate weight gain, at least 72 hours after diuretic administration

3. Diuretic administration: Administration and continued standard use of at least one agent for ³ 1 month 4. Presence of features attributable to clinical ascites at time of inappropriate weight gain—physical exam findings

and/or ultrasonographic findings of free fluid in the abdomen

(3)

- Possible clinically-evident ascites (pCE ascites): Clinical diagnosis of ascites with institution of chronic diuretic therapy³

Development of ascites has been reported as an important sentinel event in various pediatric chronic liver disease studies (3, 16-24). However, there remains no consensus criteria for diagnosis of clinically evident ascites in pediatric CLD. Historically, ascites in children has been identified through various methods, as expert reviews and studies have cited use of any combination of physical exam findings, evidence of intraabdominal fluid on imaging, and/or associated inappropriate weight gain for diagnosis. However, isolated use of these various diagnostic techniques remains fraught with variable confounders and are subject to a range of varying sensitivities/specificities (25, 26).

In this study, the primary criterion for our definition of clinically evident ascites was the documented evidence of inappropriate weight gain, with subsequent response to diuretic administration by evidence of weight loss. Historically, inappropriate weight gain has been identified as a clinical indication of developing pediatric ascites and should be appreciable in the setting of clinically evident ascites (1, 19, 27). Furthermore, an appropriate response of weight loss to diuretic administration (16) should further implicate the presence of true clinical ascites (versus organomegaly which may not result in rapid weight gain or loss). It is

acknowledged that variables such as organomegaly or anasarca may confound weight gain in infants with BA (3, 5). Thus, a cutoff of >95^th percentile in weight gain velocity (essentially, 2 standard deviations) was implemented to circumvent these variables. Anasarca is typically a late event in the context of hypoalbuminemia and/or hyponatremia. As such, it was presumed that clinically evident ascites would precede the development of this late complication of CLD.

Unfortunately, measurements of weights in children are not always reliable, as confounders such as the use of different scales, varying times of when the child is weighed, and the presence or absence of clothing at time of measurement may all skew documented weights. As such, the primary criterion of inappropriate weight gain should be confirmed by other methods of

diagnosis. Various prior expert reviews and studies have used physical exam techniques/clinical findings (i.e. bulging flanks, fluid wave, respiratory compromise in the setting large abdominal distention) and/or imaging findings suggestive of free abdominal fluid to diagnosis ascites in children (19-24). Therefore, complimentary evidence of such findings (i.e. physical exam, ultrasound free abdominal fluid) found in conjunction to an inappropriate weight gain should provide sufficient evidence for clinically evident ascites in a child.

Varices

- Clinically evident varices: Documented finding of esophageal and/or gastric varices on endoscopic evaluation

(4)

The development of varices has been recognized as a major complication of clinically significant portal hypertension in patients with cirrhosis and a sentinel marker of disease stage

progression in adults with cirrhosis (28, 29). The clinical practice of screening and primary prophylactic treatment of varices in children with CLD remains controversial (30-32). As such, there may be under-identification of varices in centers that do not follow this clinical practice (11).

Synthetic Liver Dysfunction Research Definition:

- Definite clinically evident synthetic liver dysfunction (dCE SLD): Meets criteria 1 AND 2 - Possible clinically evident synthetic liver dysfunction (pCE SLD): Only meets criteria 1

1. INR >1.5 that does not correct with intramuscular or intravenous phytonadione (Vitamin K) of age appropriate/weight-based dosing OR documented evidence of low factor V level (<40%)

2. Without evidence of disseminated intravascular coagulation (DIC)

In this study, synthetic liver dysfunction (SLD) was defined by the evidence of significant coagulopathy in the absence of DIC. An INR of >1.5 was chosen as a diagnostic criterion, as this cutoff has historically been included in the diagnosis of pediatric acute liver failure (33) and in the Asian-Pacific (APASL) definition of pediatric acute-on-chronic liver failure(34-36). In general, synthetic liver dysfunction (SLD) is thought to typically manifest in the latter stages of disease course in patients with BA and may be mistakenly diagnosed earlier on in the setting of the alternative explanation of vitamin K deficiency secondary to cholestasis(1).

Recurring Clinical Events Variceal Hemorrhage Research Definition

- Clinically evident variceal hemorrhage: Documented gastrointestinal hemorrhage with endoscopic evidence of esophageal and/or gastric varices in the absence of any other source of hemorrhage

For this study, variceal hemorrhage was defined separately from varices and designated as a recurring clinical event, as it occurs only in the setting of established varices and may present multiple times throughout the disease course. We acknowledge that the identification of varices and variceal hemorrhage may be concurrent by these criteria but emphasize that the definitions are for purposes of clinical evidence of manifestations and/or events. Endoscopic requirement for diagnosis was set in order to avoid mistaking other causes of hematemesis in children, such as Mallory-Weiss tears, gastritis, or swallowed blood secondary to epistaxis (37, 38), from true variceal hemorrhage.

(5)

Cholangitis

- Probable clinically evident cholangitis (A) 1. Fever (>38.0 C)

2. An increase ^{a, b} in conjugated bilirubin from baseline¹

1. Baseline: Average of 3 consecutive conjugated bilirubin measurements prior to increase a. If conjugated bilirubin baseline <1.5 mg/dL, then increase defined as >0.3 mg/dL

I. If conjugated bilirubin assay unavailable: if direct bilirubin baseline <1.5 mg/dL, then increase defined as >0.5 mg/dL*

b. If conjugated bilirubin baseline >1.5 mg/dL, then increase defined as >20%

I. If conjugated bilirubin assay unavailable: if direct bilirubin baseline >1.5 mg/dL, then increase defined as >30%*

*recommendations for direct bilirubin are empiric and not based upon the associated published data that relies on changes in conjugated bilirubin levels

3. Afebrile within 72 hours of initiation of antibiotic treatment 4. Without evidence of an alternative source of infection

5. Documented clinical attribution of cholangitis as rationale for intravenous antibiotic treatment for at least 7 days

- Presumed clinically evident cholangitis (B) 1. Fever (>38.0 C)

2. Does not meet criteria for increase in conjugated bilirubin from baseline 3. Afebrile within 72 hours of initiation of antibiotic treatment

4. Without evidence of an alternative source of infection

- Possible clinically evident cholangitis (C) 1. Fever (>38.0 C)

2. Increase in conjugated bilirubin from baseline 3. Afebrile within 72 hours of antibiotic treatment

4. Patient is documented positive for another source of infection

Cholangitis is commonly identified as a major infectious complication in the disease course of BA, but remains without a definitive, consensus pediatric definition (1). Given the variability that currently exists for diagnostic criteria of cholangitis in both research investigations and in clinical practice, three variations of definition were developed for purposes of a broad

identification in our cohort. Diagnostic criteria were developed from a review of existing non- consensus definitions published in pediatric literature and expert opinion review (1, 39-49).

Evidence of bacteremia was not included as a criterion, as previous reports demonstrate <50%

positive blood cultures in children diagnosed with cholangitis (1, 41, 44). For all three variations of the cholangitis definition, diagnosis required development of fever and response to >7 days

(6)

of antibiotic therapy (by reduction of temperature <38.0 C), with specific clinical documentation of attribution of cholangitis as the main clinical reason for long-term intravenous antibiotic treatment.

“Jaundice” is commonly cited as a general criterion for cholangitis. However, we are unaware of published pediatric cutoffs for elevation of bilirubin to define “jaundice.” Therefore, specific rigorous cutoffs for conjugated bilirubin elevation were implemented to define “jaundice” in both the “probable” and “possible” definitions. Conjugated bilirubin was selected as the primary assay to diagnosis jaundice for two main reasons. First, these operational definitions and their testing were developed at our institution where direct bilirubin is not measured.

Second, conjugated bilirubin has historically been judged to be a more accurate marker for cholestasis (50) and should be used instead of direct bilirubin as a criterion for jaundice at institutions where the conjugated assay is available.

However, the exclusive use of conjugated bilirubin would severely limit the use of such

definitions, as not all pediatric centers can test for conjugated bilirubin. Thus, we proposed an alternative criterion for direct bilirubin with higher cutoffs, as direct bilirubin is known to also include a degree of delta bilirubin (and to a lesser degree, unconjugated bilirubin). These alternative definitions may potentially be tested at other institutions that primarily use direct bilirubin as a marker for cholestasis.

Overall, criteria for “probable CE cholangitis” were the most rigorous of the three variations, requiring evidence of systemic inflammation (fever) with subsequent response to antibiotic therapy (afebrile within 72 hours of treatment), evidence of jaundice (requiring elevation of conjugated or direct bilirubin levels above specific cutoffs), lack of other sources of infection, and clinical attribution of cholangitis per provider for justification for long-term IV antibiotics.

Despite being the most rigorous definition variation, the term “probable” was chosen over the term “definite”, as more direct techniques such bile aspirate collections or liver biopsy are invasive in nature and rarely performed for specific evaluation of pediatric cholangitis.

Definition variations designated “presumed CE cholangitis” (no required bilirubin increase) and

“probable CE cholangitis” (patient could be positive for other sources of infection) cholangitis were created secondary to the general variability in diagnosis of cholangitis in clinical practice.

Comparison of patients identified by these three variations of definitions may potentially be analyzed for a quality improvement sub-analysis of diagnostic and treatment practices.

Spontaneous Bacterial Peritonitis Research Definition:

- Culture-positive: Ascitic fluid culture positive for bacteria with 1) ascitic fluid

polymorphonuclear cell count >250 mm^3 or 2) ascitic fluid WBC count >500 mm^3 - Culture-negative: Ascitic fluid culture negative for bacteria with 1) ascitic fluid

polymorphonuclear cell count >250 mm^3 or 2) ascitic fluid WBC count >500 mm^3

(7)

The diagnostic criteria for spontaneous bacterial peritonitis are relatively straightforward, requiring analysis of peritoneal fluid for identification of leukocytosis with or without evidence of bacteria. Surprisingly, relatively few comprehensive pediatric publications specifically describe the prevalence and/or characteristics of this important event in BA and/or children with CLD (1, 18, 51-53).

References

1 Shneider BL, Mazariegos GV Biliary atresia: a transplant perspective. Liver Transpl 2007;13(11):1482-95.

2 Utterson EC, Shepherd RW, Sokol RJ, et al. Biliary atresia: clinical profiles, risk factors, and outcomes of 755 patients listed for liver transplantation. J Pediatr 2005;147(2):180- 5.

3 DeRusso PA, Ye W, Shepherd R, et al. Growth failure and outcomes in infants with biliary atresia: a report from the Biliary Atresia Research Consortium. Hepatology

2007;46(5):1632-8.

4 Sundaram SS, Mack CL, Feldman AG, et al. Biliary atresia: Indications and timing of liver transplantation and optimization of pretransplant care. Liver Transpl 2017;23(1):96-109.

5 Sokol RJ, Stall C Anthropometric evaluation of children with chronic liver disease. Am J Clin Nutr 1990;52(2):203-8.

6 Young S, Kwarta E, Azzam R, et al. Nutrition assessment and support in children with end-stage liver disease. Nutr Clin Pract 2013;28(3):317-29.

7 Ng VL, Haber BH, Magee JC, et al. Medical status of 219 children with biliary atresia surviving long-term with their native livers: results from a North American multicenter consortium. J Pediatr 2014;165(3):539-46.e2.

8 de Onis M, Garza C, Onyango AW, et al. [WHO growth standards for infants and young children]. Arch Pediatr 2009;16(1):47-53.

9 Protheroe SM Feeding the child with chronic liver disease. Nutrition 1998;14(10):796- 800.

10 Bass LM, Shneider BL, Henn L, et al. Clinically Evident Portal Hypertension: An

Operational Research Definition for Future Investigations in the Pediatric Population. J Pediatr Gastroenterol Nutr 2019;68(6):763-67.

11 Shneider BL, Magee JC, Karpen SJ, et al. Total Serum Bilirubin within 3 Months of Hepatoportoenterostomy Predicts Short-Term Outcomes in Biliary Atresia. J Pediatr 2016;170(211-7.e1-2.

12 Shneider BL, Abel B, Haber B, et al. Portal hypertension in children and young adults with biliary atresia. J Pediatr Gastroenterol Nutr 2012;55(5):567-73.

13 Harpavat S, Finegold MJ, Karpen SJ Patients with biliary atresia have elevated

direct/conjugated bilirubin levels shortly after birth. Pediatrics 2011;128(6):e1428-33.

(8)

14 Harpavat S, Ramraj R, Finegold MJ, et al. Newborn Direct or Conjugated Bilirubin Measurements As a Potential Screen for Biliary Atresia. J Pediatr Gastroenterol Nutr 2016;62(6):799-803.

15 Serinet MO, Wildhaber BE, Broué P, et al. Impact of age at Kasai operation on its results in late childhood and adolescence: a rational basis for biliary atresia screening.

Pediatrics 2009;123(5):1280-6.

16 Shneider BL, Brown MB, Haber B, et al. A multicenter study of the outcome of biliary atresia in the United States, 1997 to 2000. J Pediatr 2006;148(4):467-74.

17 Bezerra JA, Spino C, Magee JC, et al. Use of corticosteroids after

hepatoportoenterostomy for bile drainage in infants with biliary atresia: the START randomized clinical trial. JAMA 2014;311(17):1750-9.

18 Preto-Zamperlini M, Farhat SC, Perondi MB, et al. Elevated C-reactive protein and spontaneous bacterial peritonitis in children with chronic liver disease and ascites. J Pediatr Gastroenterol Nutr 2014;58(1):96-8.

19 Giefer MJ, Murray KF, Colletti RB Pathophysiology, diagnosis, and management of pediatric ascites. J Pediatr Gastroenterol Nutr 2011;52(5):503-13.

20 Nightingale S, Stormon MO, O'Loughlin EV, et al. Early Posthepatoportoenterostomy Predictors of Native Liver Survival in Biliary Atresia. J Pediatr Gastroenterol Nutr 2017;64(2):203-09.

21 Pugliese R, Fonseca EA, Porta G, et al. Ascites and serum sodium are markers of increased waiting list mortality in children with chronic liver failure. Hepatology 2014;59(5):1964-71.

22 Karnsakul W, Ingviya T, Seaberg E, et al. Ascites in Children: A Single-Center Experience of 27 Years. J Pediatr Gastroenterol Nutr 2017;64(1):83-88.

23 Sen Sarma M, Yachha SK, Bhatia V, et al. Safety, complications and outcome of large volume paracentesis with or without albumin therapy in children with severe ascites due to liver disease. J Hepatol 2015;63(5):1126-32.

24 Kramer RE, Sokol RJ, Yerushalmi B, et al. Large-volume paracentesis in the management of ascites in children. J Pediatr Gastroenterol Nutr 2001;33(3):245-9.

25 Cattau EL, Benjamin SB, Knuff TE, et al. The accuracy of the physical examination in the diagnosis of suspected ascites. JAMA 1982;247(8):1164-6.

26 Chongtham DS, Singh MM, Kalantri SP, et al. Accuracy of clinical manoeuvres in detection of minimal ascites. Indian J Med Sci 1998;52(11):514-20.

27 Hardy S, Kleinman RF. Cirrhosis and chronic liver failure. In: F. J. Suchy, R. J. Sokol and W.

F. Balistreri eds. Liver Disease in Children. Cambridge University Press; 2007:97-118.

28 D'Amico G, Pasta L, Morabito A, et al. Competing risks and prognostic stages of cirrhosis:

a 25-year inception cohort study of 494 patients. Aliment Pharmacol Ther 2014;39(10):1180-93.

29 D'Amico G, Morabito A, D'Amico M, et al. Clinical states of cirrhosis and competing risks.

J Hepatol 2018;68(3):563-76.

30 Wanty C, Helleputte T, Smets F, et al. Assessment of risk of bleeding from esophageal varices during management of biliary atresia in children. J Pediatr Gastroenterol Nutr 2013;56(5):537-43.

(9)

31 Shneider BL, Bosch J, de Franchis R, et al. Portal hypertension in children: expert pediatric opinion on the report of the Baveno v Consensus Workshop on Methodology of Diagnosis and Therapy in Portal Hypertension. Pediatr Transplant 2012;16(5):426-37.

32 Shneider BL, de Ville de Goyet J, Leung DH, et al. Primary prophylaxis of variceal bleeding in children and the role of MesoRex Bypass: Summary of the Baveno VI Pediatric Satellite Symposium. Hepatology 2016;63(4):1368-80.

33 Squires RH, Shneider BL, Bucuvalas J, et al. Acute liver failure in children: the first 348 patients in the pediatric acute liver failure study group. J Pediatr 2006;148(5):652-58.

34 Alam S, Lal BB, Sood V, et al. Pediatric Acute-on-Chronic Liver Failure in a Specialized Liver Unit: Prevalence, Profile, Outcome, and Predictive Factors. J Pediatr Gastroenterol Nutr 2016;63(4):400-5.

35 Lal BB, Sood V, Khanna R, et al. How to identify the need for liver transplantation in pediatric acute-on-chronic liver failure? Hepatol Int 2018;12(6):552-59.

36 Sarin SK, Kumar A, Almeida JA, et al. Acute-on-chronic liver failure: consensus recommendations of the Asian Pacific Association for the study of the liver (APASL).

Hepatol Int 2009;3(1):269-82.

37 Quak SH, Lam SK, Low PS Upper gastrointestinal endoscopy in children. Singapore Med J 1990;31(2):123-6.

38 Nasher O, Devadason D, Stewart RJ Upper Gastrointestinal Bleeding in Children: A Tertiary United Kingdom Children's Hospital Experience. Children (Basel) 2017;4(11).

39 Angelico R, Pietrobattista A, Candusso M, et al. Primary Prophylaxis for Gastrointestinal Bleeding in Children With Biliary Atresia and Portal Hypertension Candidates for Liver Transplantation: A Single-Center Experience. Transplant Proc 2019;51(1):171-78.

40 Lien TH, Bu LN, Wu JF, et al. Use of Lactobacillus casei rhamnosus to Prevent Cholangitis in Biliary Atresia After Kasai Operation. J Pediatr Gastroenterol Nutr 2015;60(5):654-8.

41 Lee JY, Lim LT, Quak SH, et al. Cholangitis in children with biliary atresia: health-care resource utilisation. J Paediatr Child Health 2014;50(3):196-201.

42 Kinney TP Management of ascending cholangitis. Gastrointest Endosc Clin N Am 2007;17(2):289-306, vi.

43 Lally KP, Kanegaye J, Matsumura M, et al. Perioperative factors affecting the outcome following repair of biliary atresia. Pediatrics 1989;83(5):723-6.

44 Wu ET, Chen HL, Ni YH, et al. Bacterial cholangitis in patients with biliary atresia: impact on short-term outcome. Pediatr Surg Int 2001;17(5-6):390-5.

45 Bu LN, Chen HL, Chang CJ, et al. Prophylactic oral antibiotics in prevention of recurrent cholangitis after the Kasai portoenterostomy. J Pediatr Surg 2003;38(4):590-3.

46 Feldman AG, Mack CL Biliary Atresia: Clinical Lessons Learned. J Pediatr Gastroenterol Nutr 2015;61(2):167-75.

47 Haber BA, Russo P Biliary atresia. Gastroenterol Clin North Am 2003;32(3):891-911.

48 Hung PY, Chen CC, Chen WJ, et al. Long-term prognosis of patients with biliary atresia: a 25 year summary. J Pediatr Gastroenterol Nutr 2006;42(2):190-5.

49 Ernest van Heurn LW, Saing H, Tam PK Cholangitis after hepatic portoenterostomy for biliary atresia: a multivariate analysis of risk factors. J Pediatr 2003;142(5):566-71.

50 Ou CN, Buffone GJ, Herr Calomeni PJ, et al. Conjugated bilirubin versus direct bilirubin in neonates. Am J Clin Pathol 1986;85(5):613-6.

(10)

51 Carey RG, Bucuvalas JC, Balistreri WF, et al. Hyponatremia increases mortality in

pediatric patients listed for liver transplantation. Pediatr Transplant 2010;14(1):115-20.

52 Srivastava A, Malik R, Bolia R, et al. Prevalence, Clinical Profile, and Outcome of Ascitic Fluid Infection in Children With Liver Disease. J Pediatr Gastroenterol Nutr

2017;64(2):194-99.

53 Vieira SMG, Schwengber FP, Melere M, et al. The first episode of spontaneous bacterial peritonitis is a threat event in children with end-stage liver disease. Eur J Gastroenterol Hepatol 2018;30(3):323-27.