Utilization of Reflex Testing for Direct Bilirubin in the Early Recognition of Biliary Atresia

Transcription

1 Clinical Chemistry 63: (2017) Evidence-Based Medicine and Test Utilization Utilization of Reflex Testing for Direct Bilirubin in the Early Recognition of Biliary Atresia Leo Lam, 1 Samarina Musaad, 2 Campbell Kyle, 1,2* and Stephen Mouat 3 BACKGROUND: Delayed diagnosis of biliary atresia is an important cause of pediatric end-stage liver failure and liver transplantation. We sought to determine whether direct bilirubin is underutilized by retrospectively reviewing patients with biliary atresia. Further, we aimed to determine the role of reflex testing for direct bilirubin in patients suspected for jaundice. METHODS: The time intervals between total bilirubin and direct bilirubin measurements were retrospectively reviewed in patients with biliary atresia. We also audited the results of two major laboratories that had implemented reflex testing for direct bilirubin. We evaluated the clinical impact and cost of reflex testing in infants with increased direct bilirubin ( 1.5 mg/dl; 25 mol/l). RESULTS: In patients with known biliary atresia, an isolated total bilirubin measurement preceded direct bilirubin measurement in 46% (40/87) of patients; with a median delay of 19 days (interquartile range 3 44 days). In the community setting, direct bilirubin had a higher clinical specificity for biliary atresia than in the hospital setting. Reporting direct bilirubin results in 1591 infants younger than 2 weeks of age in the community was associated with three admissions to the hospital, one of whom was diagnosed with biliary atresia. The cost for the two laboratories for direct-bilirubin testing was estimated at US$3200 (NZ$4600 ) for each newly diagnosed case of biliary atresia. CONCLUSIONS: We identified underutilization of direct bilirubin as a cause of delay in the recognition of biliary atresia and show that reflex testing for direct bilirubin in jaundiced infants is a cost-effective solution American Association for Clinical Chemistry Biliary atresia is characterized by progressive obstruction of the extrahepatic biliary tree in the newborn. Except for jaundice and acholic stools, infants appear well during the early stages of disease, which leads to delay in diagnosis. Biliary atresia is the most common cause of pediatric end-stage liver disease and is the leading indication for pediatric liver transplantation (1, 2). The standard of care is surgical drainage of the biliary tree (Kasai operation) followed by liver transplantation for those patients who progress to end-stage liver disease. With these treatments, the reported overall survival rates are 80% 90% at 4 years (3, 4); however, if untreated, this disease leads to end-stage liver failure and death by the age of 3 years (5, 6). The age at which Kasai operation is performed is a key prognostic factor and delayed presentation is the primary reason cited for not proceeding with surgical drainage (4, 7). A continuous association has been identified between the age at which Kasai operation is performed and long-term outcome (5). This association has also been made in countries that have actively reduced delays to Kasai operation using a stool color card screening program (8, 9). Taken together, earlier recognition of biliary atresia is a modifiable factor that improves clinical outcome. In this regard, multiple investigators have raised the importance of evaluating direct or conjugated bilirubin (DB/CB) 4 for biliary atresia in newborn screening (10 14). Studies to date suggest that a high clinical sensitivity and specificity could be achieved using DB/CB in infants from birth to 60 h of life (10, 11). Although further evaluation of newborn screening by DB/CB is still required, a potential problem is the collection of an additional plasma sample, which is currently not routinely performed for newborn screening in many countries. In this study, we sought to determine if automated requests initiated by clinical chemistry analyzers (reflex testing) for DB/CB in infants with increased total bilirubin could be a cost-effective practice in identifying those with biliary atresia. We achieved this by retrospectively reviewing a cohort of patients with biliary atresia to determine if there was a significant delay in requesting DB/ CB, after total bilirubin was tested. In addition, we de- 1 Department of Chemical Pathology, Labplus, Auckland City Hospital, Auckland, New Zealand; 2 Department of Biochemistry, Labtests, Auckland, New Zealand; 3 Department of Paediatric Gastroenterology, Starship Hospital, Auckland, New Zealand. * Address correspondence to this author at: LabPlus, Auckland City Hospital, Auckland, New Zealand. Fax ; CampbellK@adhb.govt.nz. Received October 24, 2016; accepted January 11, Previously published online at DOI: /clinchem American Association for Clinical Chemistry 4 Nonstandard abbreviations: DB/CB, direct bilirubin or conjugated bilirubin; ISO, International Organization for Standardization; CRAN, Comprehensive R Archive Network. 973

2 termined the cost and impact on hospital services using data from two laboratories that provide services for different populations: infants from the community and the hospital. Methods IDENTIFICATION OF PATIENTS Laboratory and clinical information from 3 separate cohorts were examined: patients with biliary atresia, patients with suspected jaundice in the community, and patients tested at a tertiary/quaternary referral hospital. A cohort of patients with biliary atresia was identified across New Zealand from a national database of all children with serious gastrohepatic disease. This database is managed by the Department of Gastroenterology at Starship Hospital, New Zealand. All patients with biliary atresia born in New Zealand from January 2003 to April 2016 were included. Biliary atresia was diagnosed by intraoperative cholangiogram or identification of an atretic extrahepatic biliary tree at the time of the Kasai operation. Where the Kasai operation was contraindicated, the diagnosis of biliary atresia was established in the presence of supportive histological findings and exclusion of other causes of liver failure such as Alagille syndrome, progressive familial intrahepatic cholestasis, and -1 antitrypsin deficiency. In total, 88 patients were identified and complete medical/laboratory records were obtained in 87 patients. One patient was excluded because laboratory records could not be obtained due to a change in laboratory provider and laboratory information system. Laboratory results and clinical information for each patient were requested from the patient s domicile laboratory and hospital. In total, 4030 consecutive results for total bilirubin were obtained from the community laboratory (Labtests Auckland) in infants younger than 6 months old, and 2608 consecutive results in infants between 2 weeks and 6 months old were obtained from the hospital laboratory (LabPlus, Auckland City Hospital) for the period between May 2014 and September 2015 inclusive. The community laboratory provides testing services for infants in Auckland in the community setting, while the hospital laboratory provides services for inpatients and some outpatients at the National Women s Hospital and the Starship Children s Hospital. These hospitals are tertiary/quaternary referral centers and national service providers for patients in the pediatric intensive care unit, liver transplant unit, heart transplant unit, metabolic services, and all levels of neonatal intensive care. For each patient with increased DB/CB direct bilirubin, electronic clinical records were retrieved to identify the cause. Where the same patient was tested at both laboratories, the earliest result was used. The cause of increased direct bilirubin was categorized for each patient (Table 1). Table 1. Classification of patients with increased direct bilirubin. a Category New biliary atresia Gastrohepatic PICU/NICU b Hypothyroidism Unexplained Antibody-mediated hemolysis New cases of biliary atresia. All patients with a primary or secondary gastrointestinal or hepatic cause for increased direct bilirubin. These causes include biliary atresia (existing cases), intestinal failure associated liver disease, neonatal hepatitis, giant cell hepatitis, paucity of biliary ducts or Alagille syndrome, liver failure from type 1 tyrosinemia, or of uncertain etiology and intrahepatic hemangioma. Any patients admitted to an intensive care unit or are critically ill. These patients have multifactorial causes for jaundice. Where no other causes could be identified except for hypothyroidism. All patients investigated for jaundice for which there was no cause identified or where no further followup was required. All patients with positive direct antiglobulin test where an alternative cause could not be found. a Patients with increased bilirubin were categorized based on review of their clinical documentation. b PICU/NICU, pediatric intensive care unit/neonatal intensive care unit. The study received institutional approval from the Auckland District Health Board (A 6619) and was exempted from further review by the New Zealand Health and Disability Ethics Committees. LABORATORY METHODS AND CRITERIA FOR REFLEX TESTING In the community laboratory, reflex testing for direct bilirubin was performed on all children younger than 6 months of age. The same was performed in the hospital laboratory for children between 2 weeks and 6 months old with a total bilirubin 3.5 mg/dl ( 60 mol/l). At both laboratories, direct bilirubin was performed using the Roche method for direct bilirubin (generation 2) on a Cobas c501 analyzer. Total bilirubin was measured using the Roche method for total bilirubin (generation 3) on a Cobas c501 analyzer at the community laboratory and 974 Clinical Chemistry 63:5 (2017)

3 Reflex Testing for Direct Bilirubin Table 2. Timing of bilirubin tests and Kasai operations in patients with biliary atresia. a Simultaneous (n = 47) Delayed (n = 40) P value Age at first total bilirubin, days 49 (33 64) 6 (2 23) < b Age at first DB/CB, days 46 (34 63) 44 (15 42) 0.17 Age at Kasai, days 63 (50 70) 60 (43 75) 0.49 Delay between DB/CB and Kasai, days 13 (9 19) 19 (13 27) a Patients with biliary atresia were separated into patients who had DB/CB performed at the same time (Simultaneous) and those that did not (Delayed). Delays are recorded in days with the interquartile range shown in parentheses. Two patients who did not receive Kasai operation were excluded for Kasai related measurements. b Bonferroni adjusted P value of < indicated a significant difference. the same assay on a Cobas c701 analyzer at the hospital laboratory. Samples tested for direct bilirubin were identified as hemolyzed using an H-index cutoff of 25, corresponding to an approximate plasma hemoglobin concentration of 25 mg/dl (15.5 mol/l) at both laboratories. Both laboratories were accredited by International Accreditation New Zealand under the International Organization for Standardization (ISO)15189 standard for laboratories and maintain excellent performance in the Royal College of Pathologists of Australasia external Quality Assurance Program for these analytes. STATISTICS AND CALCULATIONS Comparison of the timing of tests and the Kasai operation was performed using the Mann Whitney U-test. Calculation of clinical specificity was performed by identifying results from patients with a new diagnosis of biliary atresia as positive cases and all remaining cases that fulfilled the reflex criteria for each laboratory as negative. A distribution of specificities was calculated by bootstrap resampling with 2000 repetitions using the proc package from the Comprehensive R Archive Network (CRAN) (15). The clinical specificity and its confidence interval were taken from the median, 2.5th and 97.5th percentile of this distribution. In patients with more than one direct bilirubin result, we took the first direct bilirubin that fulfilled the criteria of reflex testing for each laboratory. For both laboratories, the estimated cost for each direct bilirubin test was US$0.70 (NZ$1) factoring in reagent cost, labor, and fixed overheads. The overall cost per new diagnosis was calculated by following formula: (cost per direct bilirubin test) (number of direct bilirubin tests performed)/(number of new biliary atresia identified by reflex). Results UNDERUTILIZATION AND DELAY IN TESTING OF DIRECT BILIRUBIN Examining the national database for biliary atresia, of 87 patients identified with biliary atresia, 47 had testing for both DB/CB and total bilirubin on their initial sample while 40 patients did not have testing done. The only significant difference between the 2 groups was the age at which total bilirubin was tested (Table 2). Interestingly, the age at which the Kasai operation was performed did not reach statistical significance between the two groups. For patients who did not have simultaneous testing for DB/CB, 26 patients were younger than 14 days of age at the time of their first total bilirubin test. The age at which Kasai operation was performed correlated with the age of the first DB/CB test (R 0.85, P ). There was a weak association with age at the time of first total bilirubin test (R 0.49, P ). In patients where initial simultaneous testing was not performed, the median time before DB/CB testing was 19 days (interquartile range 3 44, Fig. 1). These findings suggested that children with biliary atresia often had total bilirubin tested at an early age. Thus, the opportunity to recognize this disease was often missed due to the underutilization of DB/CB. POTENTIAL BENEFIT OF REFLEX TESTING Examining the two laboratories that had recently implemented reflex testing, hemolysis and insufficient sample volume were identified as common causes for the inability to test for DB/CB. These causes affected 18% of samples fulfilling the reflex criteria in the community and 29% in the hospital setting (Fig. 2). In examining the first DB/CB result for each patient that fulfilled the reflex criteria, the median age at the time of first total bilirubin test was found to be 8 days (interquartile range 4 24) in the community and 27 days (interquartile range 20 40) in the hospital. Using a clinical decision-making threshold of 1.5 mg/dl (25 mol/l) for DB/CB, the clinical specificity was 99.1% (95% CI 98.7% 99.5%) in the community and 69% (61% 77%) at the hospital. Table 1 in the Data Supplement that accompanies the online version of this article at 63/issue 5 shows a contingency table for patients who fulfilled the criteria for reflex testing. At every cutpoint examined, the clinical specificity remained higher Clinical Chemistry 63:5 (2017) 975

4 in the community setting after accounting for the difference in age (see online Supplemental Fig. 1). Since we were unable to determine from the laboratory information systems which of the samples had undergone reflex testing, request forms for patients with increased DB/CB were examined to determine whether direct bilirubin had been requested by clinicians. In taking the first increased direct bilirubin result for each patient, reflex testing identified 29% (10/34) of community patients and 59% (26/44) of hospital patients with increased DB/CB. These findings indicated that reflex testing identified a large proportion of patients with increased DB/CB who would have been missed by relying on clinical requesting. Fig. 1. Delay in obtaining direct or conjugated bilirubin. The delay between obtaining total bilirubin and the first DB/CB in infants with biliary atresia that did not have simultaneous testing. No significant difference could be observed between those younger or older than 14 days of age (P = 0.12). CLINICAL IMPACT AND COST OF REFLEX TESTING The most common association with increased DB/CB in patients in the community was antibody-mediated hemolysis, although most remained unexplained (Table 3); these patients had a relatively low proportion of DB/CB compared to their total bilirubin (Fig. 3). In contrast, gastrohepatic disorders were the most common cause for increased DB/CB in the hospital. In keeping with local and international guidelines, all patients in the community older than two weeks of age with increased DB/CB, except one, were followed up at the hospital. In contrast, DB/CB testing in 1591 infants younger than 2 weeks resulted in 21 patients with increased DB/CD and three admissions to hospital. In two of these, no cause for jaundice could be identified, while a new diagnosis of biliary atresia was made in one. For the remaining 18 infants investigated in the community, 16 had follow-up, including repeat total and direct bilirubin, which was considered reassuring; however, none of them progressed to imaging as a final rule-out investiga- Fig. 2. Requests for total bilirubin from the community and hospital laboratory. The number of samples and individuals less than 2 weeks of age is indicated in parentheses. A direct bilirubin of 1.5 mg/dl corresponds to 25 μmol/l. 976 Clinical Chemistry 63:5 (2017)

5 Reflex Testing for Direct Bilirubin Table 3. Number of patients with increased direct bilirubin detected by reflex testing. a Overall Community N Age, days 14 days >14 days Hospital, >14 days New biliary atresia 4 37 (14 65) Gastrohepatic (24 83) PICU/NICU b (16 62) Hypothyroidism 2 64 (63 66) Antibody-mediated hemolysis 7 4 ( ) Unexplained 17 5 (4 20) Total (6 61) a Where a patient had more than 1 test from both laboratories, only the first increased direct bilirubin was accounted for. The median age and interquartile ranges of patients are provided. b Three neonates were tested for direct bilirubin in the community laboratory within 5 days of discharge from neonatal intensive care unit (NICU). These neonates either did not fulfil reflex criteria of the hospital laboratory or were treated at a different hospital. PICU/NICU, pediatric intensive care unit/nicu. tion. These findings suggested that, in patients under 2 weeks of age, reflex testing for DB/CB was useful in identifying patients with biliary atresia and did not lead to a substantial increase in the number of hospital presentations. During the study period, a total of 8 patients were identified as having new or preexisting biliary atresia, of which four had a new diagnosis. One of the four was identified as having increased direct bilirubin via reflex testing (as previously described); in the remaining 3 patients, direct bilirubin was requested by a clinician. Using each laboratory s criteria for reflex testing on all samples received, the cost of DB/CB tests combined for both community and hospital laboratories over a period of 17 months was estimated at US$3200 (NZ$4600 ) for each new case of biliary atresia identified. Discussion In this study, we investigated the value of testing for DB/CB in addition to total bilirubin in infants investi- Fig. 3. Distribution of total, direct, and percentage direct bilirubin in patients with increased direct bilirubin detected by reflex testing. Patients with increased direct bilirubin were categorized according to their underlying medical illness. The median, interquartile range, and absolute range are shown for each category. PICU/NICU, pediatric intensive care unit/neonatal intensive care unit; AMH, antibody-mediated hemolysis. Clinical Chemistry 63:5 (2017) 977

6 gated for jaundice. We examined three separate datasets: the national database for biliary atresia and datasets from a community laboratory and a hospital laboratory that had implemented reflex testing. Although previous studies have indicated that a high clinical sensitivity and specificity of DB/CB during the first days of life can be used to identify biliary atresia (10, 11, 16), the implementation of a national screening program for biliary atresia by testing for DB/CB has not been fully evaluated (13). Reflex testing therefore represents an opportunity to detect patients with biliary atresia at an earlier age. We found that the timing of DB/CB testing was associated with the age at which the Kasai operation was performed. However, this test was often requested later than total bilirubin. This is supported by our retrospective audit of patients with increased DB/CB, where a large proportion of requesters did not indicate DB/CB on their request forms. Taken together, our results indicate delays in obtaining DB/CB as an important cause of delay in the diagnosis of biliary atresia. Reflex testing is an operator independent mechanism that identifies all patients with potentially increased direct bilirubin. This test has high clinical specificity in the community, while in the hospital clinical specificity is lower because of associated morbidities, e.g., critical illness and gastrohepatic pathologies. The consequence of delayed diagnosis and management of biliary atresia is a reduction in native liver survival or overall survival (5). At our center, a case of pediatric liver transplant and its associated cost is estimated at US$ (NZ$250000) with an ongoing annual cost of US$14000 (NZ$20000) per year. In comparison, the estimated cost of US$3200 (NZ$4600) per newly diagnosed case of biliary atresia supports reflex testing for DB/CB as highly cost-effective. Furthermore, testing DB/CB on all patients in the community in the past 17 months did not result in a substantial increase in the number of patients unnecessarily presenting to the hospital. These findings suggest that reflex testing is a costeffective intervention with minimal interruption to hospital service delivery. The strengths of our study include the availability of a database containing all patients in New Zealand with biliary atresia over a 13-year period, which allowed us to identify baseline practices of all laboratories in the country, as well as all patients with biliary atresia who had reflex testing. Because the same DB/CB assay was used in the community and hospital laboratories, a fair comparison of results could be made. In contrast to population screening, reflex testing has the advantage of identifying cases among children who present to laboratories for blood tests and therefore are more likely to be clinically jaundiced. DB/CB testing in this population has a higher pretest probability for biliary atresia. In our study, 4 of 2312 patients were identified with newly diagnosed biliary atresia, a considerably higher rate than the typical rate of 1: 8000 in New Zealand derived from our national database and the 1: : reported in some European countries (3). The higher pretest probability results in a lower cost of performing this test for each new diagnosis made. However, it should be noted that reflex testing alone does not improve the time to first presentation to the healthcare system and is therefore not a complete substitute for a national screening program. Although current guidelines recommend the testing of DB/CB in all infants suspected of jaundice who are older than 2 weeks (17, 18), our findings indicate nonadherence to this recommendation as another cause of delayed management in children with biliary atresia. One limitation of reflex testing is providing results that clinicians may not anticipate, which may lead to inadequate follow-up or management. In this regard, the hospital laboratory has adopted automated commenting advising clinical staff to consider the causes of increased DB/CB and to monitor for acholic stools. In addition, the community laboratory regards all DB/CB results 1.5 mg/dl ( 25 mol/l) in infants as critical, requiring communication to the clinician by telephone. However, we were unable to determine if all patients with increased direct bilirubin were followed up appropriately. The limitations of our study include the relatively small number of newly diagnosed biliary atresia patients, which limited the ability to determine the diagnostic utility of DB/CB testing. In addition, current DB/CB measurements across different analyzers are not harmonized (19, 20), and there is a lack of consensus on the appropriate clinical decision making threshold. The use of reflex DB/CB testing in hospital patients 14 days of age was not evaluated in our study because the hospital laboratory had not implemented reflex testing. Finally, the limited number of newly diagnosed biliary atresia patients did not allow the measurement of clinical outcomes. Our results indicate that delays in DB/CB testing occur in a large proportion of children with biliary atresia and support reflex testing as a cost-effective solution to overcome this problem. Laboratories should consider differences between patients in the community and hospital when implementing reflex testing and consider increased direct bilirubin as a critical test in infants from the community. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. 978 Clinical Chemistry 63:5 (2017)

7 Reflex Testing for Direct Bilirubin Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: C.V. Kyle, Labtests Auckland. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. References Patents: None declared. Role of Sponsor: No sponsor was declared. Acknowledgments: We would like to thank members of the Auckland Regional Quality Assurance Group for their support and implementation of reflex testing for direct/conjugated bilirubin. We would like to thank Associate Professor James Davidson (former head of Chemical Pathology, LabPlus) for his assistance with the proofreading of the manuscript. We wish him all the best in his retirement. 1. Wilde J, Chin S, Johnston P, McCall J, Munn S, Nixon C, et al. Paediatric liver transplantation in New Zealand: the first 5 years. N Z Med J 2007;120:U Sokol RJ, Mack C, Narkewicz MR, Karrer FM. Pathogenesis and outcome of biliary atresia: Current concepts. J Pediatr Gastroenterol Nutr 2003;37: Chardot C. Biliary atresia. Orphanet J Rare Dis 2006;1: Schreiber RA, Barker CC, Roberts EA, Martin SR, Alvarez F, Smith L, et al. Biliary atresia: the Canadian experience. J Pediatr 2007;151:659 65, 665.e1. 5. Serinet MO, Wildhaber BE, Broue P, Lachaux A, Sarles J, Jacquemin E, 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: Bates MD, Bucuvalas JC, Alonso MH, Ryckman FC. Biliary atresia: pathogenesis and treatment. Semin Liver Dis 1998;18: Superina R, Magee JC, Brandt ML, Healey PJ, Tiao G, Ryckman F, et al. The anatomic pattern of biliary atresia identified at time of Kasai hepatoportoenterostomy and early postoperative clearance of jaundice are significant predictors of transplant-free survival. Ann Surg 2011; 254: Gu YH, Yokoyama K, Mizuta K, Tsuchioka T, Kudo T, Sasaki H, et al. Stool color card screening for early detection of biliary atresia and long-term native liver survival: a 19-year cohort study in Japan. J Pediatr 2015;166: e1. 9. Lien TH, Chang MH, Wu JF, Chen HL, Lee HC, Chen AC, et al. Effects of the infant stool color card screening program on 5-year outcome of biliary atresia in Taiwan. Hepatology 2011;53: Harpavat S, Finegold MJ, Karpen SJ. Patients with biliary atresia have elevated direct/conjugated bilirubin levels shortly after birth. Pediatrics 2011;128:e Harpavat S, Ramraj R, Finegold MJ, Brandt ML, Hertel PM, Fallon SC, et al. Newborn direct/conjugated bilirubin measurements as a potential screen for biliary atresia. J Pediatr Gastroenterol Nutr 2015;62: Wang KS, Section on Surgery, Committee on Fetus and Newborn, Childhood Liver Disease Research Network. Newborn screening for biliary atresia. Pediatrics 2015; 136:e Sokol RJ. Newborn screening for biliary atresia: bilirubin or bust? J Pediatr Gastroenterol Nutr 2016;63: Sokol RJ, Shepherd RW, Superina R, Bezerra JA, Robuck P, Hoofnagle JH. Screening and outcomes in biliary atresia: summary of a National Institutes of Health workshop. Hepatology 2007;46: Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez JC, Muller M. proc: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics 2011;12: Powell JE, Keffler S, Kelly DA, Green A. Population screening for neonatal liver disease: potential for a community-based programme. J Med Screen 2003; 10: MoyerV, FreeseDK, WhitingtonPF, OlsonAD, BrewerF, Colletti RB, Heyman MB. Guideline for the evaluation of cholestatic jaundice in infants: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2004;39: Fawaz R, Baumann U, Ekong U, Fischler B, Hadzic N, Mack CL, et al. Guideline for the evaluation of cholestatic jaundice in infants: joint recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, andnutritionandtheeuropeansocietyfor Pediatric Gastroenterology, Hepatology, and Nutrition. J Pediatr Gastroenterol Nutr 2017;64: Greene DN, Liang J, Holmes DT, Resch A, Lorey TS. Neonatal total bilirubin measurements: still room for harmonization. Clin Biochem 2014;47: Harpavat S, Devaraj S, Finegold MJ. An infant with persistent jaundice and a normal newborn direct bilirubin measurement. Clin Chem 2015;61: Clinical Chemistry 63:5 (2017) 979