Academy of Diagnostics & Laboratory Medicine - Scientific Short
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Bilirubin, a heme metabolite, can be toxic when it accumulates. The body has developed mechanisms to metabolize and excrete bilirubin efficiently. Initially, heme degradation produces unconjugated bilirubin (Bu), which is water-insoluble and binds tightly to albumin via non-covalent interactions, facilitating its transport to the liver. In the liver, Bu undergoes glucuronidation, forming mono- or di-glucuronide conjugates known as conjugated bilirubin (Bc). A fraction of Bc can bind albumin covalently, resulting in delta bilirubin (DBili). Due to its association with albumin, delta bilirubin has a longer half-life (12-14 days) compared to free Bc (2-4 hours).
Bilirubin testing is essential for evaluating liver disease and jaundice. Hyperbilirubinemia may result from increased bilirubin production (e.g., hemolysis), impaired hepatic uptake, or defective bile excretion. Additionally, metabolic disorders such as UDP-glucuronosyltransferase deficiency can impair conjugation. Bilirubin testing is particularly important in pediatric populations, especially neonates, who are at increased risk for hyperbilirubinemia due to elevated bilirubin production at birth, immature liver enzyme systems, and high red blood cell turnover. Common differential diagnoses for neonatal jaundice include physiological jaundice, maternal-fetal blood group incompatibility, G6PD deficiency, breastfeeding-related jaundice, and inherited disorders. Excess Bu can cross the blood-brain barrier and cause kernicterus, a form of bilirubin-induced neurotoxicity. Early intervention with phototherapy or exchange transfusion can prevent this outcome, making accurate and timely bilirubin measurement essential in neonatal care.
According to the College of American Pathologists (CAP) proficiency testing participant summary, approximately 90% of laboratories use diazo-based colorimetric assays to measure total bilirubin (TBili), i.e., direct plus indirect bilirubin. When Bu is bound to albumin, it gets stabilized by hydrogen bonds that make its central methylene group inaccessible to the diazo reagent. To enable its quantification, these assays rely on accelerators (e.g., caffeine, sodium benzoate) that disrupt the strong non-covalent interactions between Bu and albumin. The terms "direct" and "indirect" bilirubin originate from reactions performed in the absence and presence of accelerators, respectively. Thus, direct bilirubin includes both conjugated and delta fractions while indirect bilirubin represents the unconjugated fraction (Figure 1). About 10% of CAP participants use multi-layer film technology, which quantifies Bu and Bc separately by binding bilirubin to a cationic mordant that produces distinct spectral characteristics. This allows measurement of Bu and Bc at two wavelengths, excluding DBili. Other methods used by clinical laboratories to quantify direct bilirubin include vanadate oxidation and bilirubin oxidase under acidic conditions. Although direct bilirubin gets preferentially oxidized under acidic conditions, a small fraction of indirect bilirubin can get oxidized as well, resulting in cross reactivity. High-performance liquid chromatography offers the most accurate fractionation to distinguish Bu, Bc, Dbili and photobilirubin isomers, but its complexity and labor demands limit routine clinical use. In addition to differences in test principles and reagents, bilirubin fractions and assays vary in susceptibility to preanalytical factors, including (1) light exposure, which can degrade bilirubin, resulting in falsely low measurements and (2) hemolysis, which can interfere with spectrophotometric readings, causing falsely elevated results.
Figure 1 Illustration of different bilirubin fraction and terminology.
In pediatric care, TBili measurement is most relevant for managing neonatal unconjugated hyperbilirubinemia. In 1999, Bhutani et al. introduced an hour-specific serum TBili nomogram to assess the risk of hyperbilirubinemia in newborns. While widely adopted in the clinical practice to assess risk and guide decisions regarding phototherapy and exchange transfusion, the nomogram is based on a region-specific cohort and does not account for the interlaboratory variability in TBili measurements, differences in bilirubin fraction contributions, or preanalytical factors affecting result accuracy.
As emphasized by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Working Group on Neonatal Bilirubin (WG-NB), the lack of standardization among bilirubin measurement methods can lead to mismanagement of neonatal hyperbilirubinemia. Undertreatment may result in long-term neurotoxicity, while overtreatment can cause unnecessary hospital stays for phototherapy or increased morbidity from exchange transfusions. Without test standardization, successful consensus guidelines on the management of different hyperbilirubinemia conditions it not attainable. To achieve standardization, collaboration with manufacturers to successfully use calibrators based in human serum that account for the different fractions is imperative.
In summary, bilirubin testing remains a “hot topic” in pediatric laboratory medicine due to its clinical importance, methodological variability, and the ongoing need for standardization. Addressing these challenges is essential to improve diagnostic accuracy, optimize patient outcomes, and ensure consistency across laboratories.
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