Research progress on methods for determining bilirubin

doi:10.3969/j.issn.1673-8640.2026.05.016

Abstract

Abstract:

Serum bilirubin plays a role in evaluating liver function and diagnosing digestive system diseases. The main methods for bilirubin determination include diazotization method，oxidation method，high-performance liquid chromatography，direct spectrophotometry，mass spectrometry analysis and transcutaneous measurement. These methods have their own advantages and disadvantages due to different principles. This review introduces the principles，characteristics and latest research progress of different bilirubin determination methods，as well as the bilirubin determination calibration system，and discusses the development needs and trends of bilirubin determination methods and standardization. With the continuous update of bilirubin determination methods，the interchangeability of inter-laboratory quality evaluation materials and the comparability between different determination systems still face a series of challenges at present.

Key words: Bilirubin, Clinical significance, Determination method, Standardization

CLC Number:

R446.1

ZHOU Xiaoyun, ZHU Jing, SHAO Wenqi, WANG Beili, PAN Baishen, GUO Wei. Research progress on methods for determining bilirubin[J]. Laboratory Medicine, 2026, 41(5): 505-512.

Figures/Tables 1

References 47

[1]	NIELSEN M H, MØRK M, MADSEN P, et al. A simplified HPLC-based method for measuring unconjugated bilirubin,bilirubin-monoglucuronide,bilirubin-diglucuronide,and delta-bilirubin in plasma with increased conjugated bilirubin[J]. Scand J Clin Lab Invest, 2023, 83(1):64-67. DOI URL
[2]	KWO P Y, COHEN S M, LIM J K. ACG clinical guideline:evaluation of abnormal liver chemistries[J]. Am J Gastroenterol, 2017,112:18-35.
[3]	COLE P G, LATHE G H, BILLING B H. The diazo reacting pigments of serum,urine and bile[J]. Biochem J, 1953, 55(319th Meeting):xiii.
[4]	GUERRA RUIZ A R, CRESPO J, LÓPEZ MARTÍNEZ R M, et al. Measurement and clinical usefulness of bilirubin in liver disease[J]. Adv Lab Med, 2021, 2(3):352-372.
[5]	HULZEBOS C V, VITEK L, CODA ZABETTA C D, et al. Diagnostic methods for neonatal hyperbilirubinemia:benefits,limitations,requirements,and novel developments[J]. Pediatr Res, 2021, 90(2):277-283. DOI
[6]	VASAVDA C, KOTHARI R, MALLA A P, et al. Bilirubin links heme metabolism to neuroprotection by scavenging superoxide[J]. Cell Chem Biol, 2019, 26(10):1450-1460. DOI PMID
[7]	TAKEI R, INOUE T, SONODA N, et al. Bilirubin reduces visceral obesity and insulin resistance by suppression of inflammatory cytokines[J]. PLoS One, 2019, 14(10):e0223302. DOI URL
[8]	VITEK L, HINDS T D Jr, STEC D E, et al. The physiology of bilirubin:health and disease equilibrium[J]. Trends Mol Med, 2023, 29(4):315-328. DOI URL
[9]	HEIRWEGH K P, FEVERY J, BLANCKAERT N. Chromatographic analysis and structure determination of biliverdins and bilirubins[J]. J Chromatogr, 1989, 496(1):1-26. DOI URL
[10]	NGASHANGVA L, BACHU V, GOSWAMI P. Development of new methods for determination of bilirubin[J]. J Pharmaceut Biomed Anal, 2019,162:272-285.
[11]	BOO N Y, CHANG Y F, LEONG Y X, et al. The point-of-care Bilistick method has very short turn-around-time and high accuracy at lower cutoff levels to predict laboratory-measured TSB[J]. Pediatr Res, 2019, 86(2):216-220. DOI
[12]	DOUMAS B T, YEIN F, PERRY B, et al. Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase[J]. Clin Chem, 1999, 45(8 Pt 1):1255-1260. DOI URL
[13]	戴波, 杨小彤, 韦忠毅. CLSI方案评价亚硝酸钠氧化法和钒酸盐氧化法检测血清总胆红素[J]. 科技视界, 2015,7:242-244.
[14]	ARIOKA M, KOYANO K, NAKAO Y, et al. Quantitative effects of bilirubin structural photoisomers on the measurement of direct bilirubin via the vanadate oxidation method[J]. Ann Clin Biochem, 2023, 60(3):177-183. DOI URL
[15]	COUGHTRIE M W, BURCHELL B, LEAKEY J E, et al. The inadequacy of perinatal glucuronidation:immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human liver microsomes[J]. Mol Pharmacol, 1988,34:729-735.
[16]	KAWAMOTO S, KOYANO K, OZAKI M, et al. Effects of bilirubin configurational photoisomers on the measurement of direct bilirubin by the vanadate oxidation method[J]. Ann Clin Biochem, 2021,58:311-317.
[17]	ZHANG C, ZHU L, ZHANG J, et al. An effective enzymatic assay for pH selectively measuring direct and total bilirubin concentration by using of CotA[J]. Biochem Biophys Res Commun, 2021,547:192-197.
[18]	ERLINGER S, ARIAS I M, DHUMEAUX D. Inherited disorders of bilirubin transport and conjugation:new insights into molecular mechanisms and consequences[J]. Gastroenterology, 2014,146:1625-1638.
[19]	SCHLEBUSCH H, AXER K, SCHNEIDER C, et al. Comparison of five routine methods with the candidate reference method for the determination of bilirubin in neonatal serum[J]. Clin Chem Lab Med, 1990,28:203-210.
[20]	QUINN K D, NGUYEN N Q, WACH M M, et al. Tandem mass spectrometry of bilin tetrapyrroles by electrospray ionization and collision-induced dissociation[J]. Rapid Commun Mass Spectrom, 2012, 26(16):1767-1775. DOI URL
[21]	OHKAWA T, NORIKURA R, YOSHIKAWA T. Rapid LC-TOFMS method for identification of binding sites of covalent acylglucuronide albumin complexes[J]. J Pharm Biomed Anal, 2003, 31(6):1167-1176. DOI URL
[22]	NAGAR G, VANDERMEER B, CAMPBELL S, et al. Reliability of transcutaneous bilirubin device sin preterm infants:a systematic review[J]. Pediatrics, 2013,132:871-881.
[23]	OKWUNDU C I, BHUTANI V K, UTHMAN O A, et al. Transcutaneous bilirubinometry for detecting jaundice in term or late preterm neonates[J]. Cochrane Database Syst Rev, 2024, 5(5):CD011060.
[24]	OKWUNDU C I, OLOWOYEYE A, UTHMAN O A, et al. Transcutaneous bilirubinometry versus total serum bilirubin measurement for newborns[J]. Cochrane Database Syst Rev, 2023, 5(5):CD012660.
[25]	TAYLOR J A, STOUT J W, DE GREEF L, et al. Use of a smartphone app to assess neonatal jaundice[J]. Pediatrics, 2017, 140(3):e20170312. DOI URL
[26]	DOUMAS B T, PERRY B W, JENDRZEJCZAK B, et al. Pitfalls in the American Monitor kit methods for determination of total and "direct" bilirubin[J]. Clin Chem, 1982, 28(11):2305-2308. PMID
[27]	SAHOO S R, HUEY-JEN HSU S, CHOU D A, et al. Surface plasmon-enhanced fluorescence and surface-enhanced Raman scattering dual-readout chip constructed with silver nanowires:label-free clinical detection of direct-bilirubin[J]. Biosens Bioelectron, 2022,213:114440.
[28]	THANGAMUTHU M, GABRIEL W E, SANTSCHI C, et al. Electrochemical sensor for bilirubin detection using screen printed electrodes functionalized with carbon nanotubes and graphene[J]. Sensors(Basel), 2018, 18(3):800.
[29]	AHMED N S, HSU C Y, MAHMOUD Z H, et al. A graphene oxide/polyaniline nanocomposite biosensor:synthesis,characterization,and electrochemical detection of bilirubin[J]. RSC Adv, 2023, 13(51):36280-36292. DOI URL
[30]	ALSHATTERI A H, OMER K M. Smartphone-based fluorescence detection of bilirubin using yellow emissive carbon dots[J]. Anal Methods, 2022, 14(17):1730-1738. DOI URL
[31]	GUO Y, WEI C, WANG R, et al. Nanomaterials for fluorescent assay of bilirubin[J]. Anal Biochem, 2023,666:115078.
[32]	LO S, JENDRZEJCZAK B, DOUMAS B T. Bovine serum-based bilirubin calibrators are inappropriate for some diazo methods[J]. Clin Chem, 2010,56:869-882.
[33]	LO S F, DOUMAS B T. The status of bilirubin measurements in U. S. Laboratories:why is accuracy elusive?[J]. Semin Perinatol, 2011,35:141-147.
[34]	HULZEBOS C V, CAMARA J E, VAN BERKEL M, et al. Bilirubin measurements in neonates:uniform neonatal treatment can only be achieved by improved standardization[J]. Clin Chem Lab Med, 2024, 62(10):1892-1903. DOI URL
[35]	Medicare, Medicaid, CLIA programs, et al. Final rule with comment period[J]. Fed Regist, 1992,57:7002-7186.
[36]	BLÁZQUEZ R, PRADA E, RICÓS C, et al. Quality analytical specifications obtained by consensus through intercomparison programs AEFA/AEBM,SEQC y SEHH[J]. Rev Calid Asist, 2015, 30(6):341-343. DOI URL
[37]	RICÓS C, PERICH C, DOMÉNECH M, et al. Variación:biológica. Revisión desde una perspectiva práctica[J]. Rev Del Lab Clin, 2010, 3(4):192-200.
[38]	KUZNIEWICZ M W, GREENE D N, WALSH E M, et al. Association between laboratory calibration of a serum bilirubin assay,neonatal bilirubin levels,and phototherapy use[J]. JAMA Pediatr, 2016, 170(6):557-561. DOI URL
[39]	OOSTENDORP M, TEN HOVE C H, VAN BERKEL M, et al. A significant increase in the incidence of neonatal hyperbilirubinemia and phototherapy treatment due to a routine change in laboratory equipment[J]. Arch Pathol Lab Med,2024,148:e40-e47.
[40]	MAILLOUX A, CORTEY A, DELATOUR V, et al. Analytical and clinical guidelines on neonatal bilirubinemia[J]. Ann Biol Clin, 2020,78:383-397.
[41]	尚才兵, 黄美芳, 刘峰, 等. Doumas参考方法对总胆红素常规检测系统的正确度评价[J]. 中国卫生检验杂志, 2017, 27(18):2654-2657.
[42]	Clinical and Laboratory Standards Institute. Measurement procedure comparison and bias estimation using patient samples[S]. EP09-A3,CLSI, 2013.
[43]	安崇文, 曹晨影, 焦莉莉, 等. 4种总胆红素检测系统的性能评价[J]. 临床检验杂志, 2014, 32(3):230-235.
[44]	王杨, 谷冬梅, 陈京山, 等. 三种常规方法检测血清胆红素结果的可比性和偏倚评估[J]. 中国实验诊断学, 2017, 21(10):1744-1747.
[45]	KIUCHI S, IHARA H, OSAWA S, et al. A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials:a comparison of synthetic delta,unconjugated,and taurine-conjugated bilirubin[J]. Ann Clin Biochem, 2021, 58(6):563-571. DOI URL
[46]	孟祥兆, 李敏, 孙江漫, 等. 两种血清总胆红素临床检测方法的偏倚分析和一致性评价[J]. 安徽医药, 2023, 27(5):925-927.
[47]	KLAUKE R, KYTZIA H J, WEBER F, et al. Reference measurement procedure for total bilirubin in serum re-evaluated and measurement uncertainty determined[J]. Clin Chim Acta, 2018,481:115-120.

方法	原理	灵敏度（检测限）	特异性	检测时间	检测成本^①	应用场景
重氮法	胆红素与重氮试剂反应生成偶氮色素，采用分光光度法测定530/598 nm处的吸光度（A）值	较高（1.7 μmol·L^-1）	中等（受溶血、维生素C等干扰）	快速（自动化，每小时数百测试）	中等偏高（8~12元）	大型医院、临床实验室常规检测（TB和DBil）
氧化法（主要为钒酸盐法）	胆红素被氧化为胆绿素，通过测定450~460 nm处的A值来定量	高（0.5 μmol·L^-1）	高（抗干扰能力强）	快速（自动化， 5~15 min）	中等（2~25元）	大型医院、临床实验室（可能存在干扰物质的样本）
HPLC法	色谱柱分离胆红素组分，采用紫外检测器定量	高（0.1~0.5 μmol·L^-1）	极高（可区分各亚型）	长（含前处理约 20~40 min）	极高（50~ 150元）	鉴定δ-Bil、光疗后胆红素异构体、药物分析、国标修订
直接分光光度法	基于胆红素在437 nm处的特征吸收峰，采用双波长消除血红蛋白干扰	较高[波长精度高（±0.3 nm），杂散光低]	低（易受血红蛋白干扰）	快速（≤5 s）	较低	实验室快速筛查，适合微量样本
质谱分析法	离子化后通过质荷比（m/z）分析，如采用LC-TOF MS^②检测胆红素-白蛋白结合位点	极高（高分辨质谱可达 0.05 μmol·L^-1）	极高（高分辨质谱通过精确质量区分胆红素异构体）	较长（5~10 min）	极高（50~ 150元，批量检测可降至30~80元）	精准检测、研究领域（如新生儿疾病筛查、代谢研究）
经皮检测法	通过皮肤反射光谱强度间接推算血清胆红素水平	较低	低（受胎龄、体重和肤色影响）	极快（≤1 min）	低（1~5元）	新生儿动态监测、基层医院、家庭随访

方法	原理	灵敏度（检测限）	特异性	检测时间	检测成本^①	应用场景
重氮法	胆红素与重氮试剂反应生成偶氮色素，采用分光光度法测定530/598 nm处的吸光度（A）值	较高（1.7 μmol·L^-1）	中等（受溶血、维生素C等干扰）	快速（自动化，每小时数百测试）	中等偏高（8~12元）	大型医院、临床实验室常规检测（TB和DBil）
氧化法（主要为钒酸盐法）	胆红素被氧化为胆绿素，通过测定450~460 nm处的A值来定量	高（0.5 μmol·L^-1）	高（抗干扰能力强）	快速（自动化， 5~15 min）	中等（2~25元）	大型医院、临床实验室（可能存在干扰物质的样本）
HPLC法	色谱柱分离胆红素组分，采用紫外检测器定量	高（0.1~0.5 μmol·L^-1）	极高（可区分各亚型）	长（含前处理约 20~40 min）	极高（50~ 150元）	鉴定δ-Bil、光疗后胆红素异构体、药物分析、国标修订
直接分光光度法	基于胆红素在437 nm处的特征吸收峰，采用双波长消除血红蛋白干扰	较高[波长精度高（±0.3 nm），杂散光低]	低（易受血红蛋白干扰）	快速（≤5 s）	较低	实验室快速筛查，适合微量样本
质谱分析法	离子化后通过质荷比（m/z）分析，如采用LC-TOF MS^②检测胆红素-白蛋白结合位点	极高（高分辨质谱可达 0.05 μmol·L^-1）	极高（高分辨质谱通过精确质量区分胆红素异构体）	较长（5~10 min）	极高（50~ 150元，批量检测可降至30~80元）	精准检测、研究领域（如新生儿疾病筛查、代谢研究）
经皮检测法	通过皮肤反射光谱强度间接推算血清胆红素水平	较低	低（受胎龄、体重和肤色影响）	极快（≤1 min）	低（1~5元）	新生儿动态监测、基层医院、家庭随访