The most widely used methods for the assay of total bilirubin in serum are based on a coupling reaction with various diazo dyes in the presence of an accelerating agent. The ‘direct’ reaction, without accelerator, gives a good estimate of the conjugated and protein bound (δ) species of bilirubin only if carefully standardized reaction conditions are used. Enzymatic methods based on bilirubin oxidase appear to give similar results, for both total and direct bilirubin, and through difficult to calibrate, spectrophotometric methods can also give reliable results for total bilirubin at the higher concentrations seen in neonates. More sophisticated HPLC or dry-slide techniques are required for the specific assay of the various conjugated and protein-bound species of bilirubin in serum.
ChowdhuryJRWolkoffAWAriasIM. Hereditary jaundice and disorders of bilirubin metabolism. In: ScriverCRBeaudetALSlyWSValleD, eds. The Metabolic Basis of Inherited Disease (6th ed.) New York: McGraw Hill1989; 1367–408.
2.
BrodersenR. Competitive binding of bilirubin and drugs to human serum albumin studied by enzymatic oxidation. J Clin Invest1974; 54: 1353–64.
3.
WuTW. Bilirubin analysis: State of the art and future prospects. Clin Biochem1984; 17: 221–9.
4.
WellsRDrewJHHammondKB. Bilirubin-binding capacity and free bilirubin concentration: Fluorescence quenching compared with peroxidase-oxidation and Sephadex-column elution techniques. Clin Chim Acta1981; 116: 69–79.
MuracaMFeveryJBlanckaertN. Relationships between serum bilirubins and production and conjugation of bilirubin. Studies in Gilbert's syndrome, Crigler-Najjar disease, hemolytic disorders, and rat models. Gastroenterology1987; 92: 309–17.
7.
RosenthalPHentonDFelberSSinatraFR. Distribution of serum bilirubin conjugates in pediatric hepatobiliary diseases. J Pediatr1987; 110: 201–5.
8.
AdachiYInufusaHYamashitaM. Human serum bilirubin fractionation in various hepatobiliary diseases by the newly developed high performance liquid chromatography. Gastroenterol Jpn1988; 23: 268–72.
9.
LevineRL. Bilirubin: Worked out years ago?Pediatrics1979; 64: 380–5.
10.
LevineRLFredericksWRRapoportSI. Entry of bilirubin into the brain due to opening of the blood-brain barrierPediatrics1982; 69: 255–255.
11.
RobinsonPJRapoportSI. Binding effect of albumin on uptake of bilirubin by brain. Pediatrics1987; 79: 553–8.
12.
WennbergRPHanceAJ. Experimental bilirubin encephalopathy: Importance of total bilirubin, protein binding, and blood-brain barrier. Pediatr Res1986; 20: 789–92.
13.
MichaelssonM.Bilirubin determination in serum and urine. Scand J Clin Lab Invest1961; 13; Suppl. 56: 1–80.
14.
EbbesenF. Determination of serum bilirubin concentration during phototherapy of newborns and in vitro: Results compared by the direct spectrometric method and the diazo method. Clin Chem1977; 23: 695–9.
15.
MalloyHTEvelynKA. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem1937; 119: 481–90.
16.
JendrassikLGrofP. Vereinfachte photometrische Methoden zur Nestimmung des Blutbilirubins. Biochem Z1938; 297: 819.
17.
BrodyJPValdesRSavoryJ. Centrifugal analyser method for total bilirubin in serum by use of diazotised 2-chloroaniline-5-sulfonic acid. Clin Chem1979; 25: 205–8.
18.
KellyAMcKennaJPMcLellandAPercyRASpoonerRJ. A bichromatic method for total bilirubin with a Centrifichem 400. Clin Chem1979; 25: 1482–4.
19.
McPhaulLKershawMTilqueDEckfeldtJH. 2,4-Dichlorobenzenediazonium-based method for total bilirubin without interference from indican in uremic sera. Clin Chem1985; 31: 1229–31.
20.
HertzHDybkaerRLauritzenM. Direct spectrometric determination of the concentration of bilirubins in serum. Scand J Clin Lab Invest.1974; 33: 215–30.
21.
WuT-WDappenGMSpaydRWSundbergMWPowersDM. The Ektachem clinical chemistry slide for simultaneous determination of unconjugated and sugar-conjugated bilirubin. Clin Chem1984; 30: 1304–9.
22.
DoumasBTWuT-WJendrzejczakB. Delta bilirubin: Absorption spectra, molar absorptivity, and reactivity in the diazo reaction. Clin Chem1987; 33: 769–74.
23.
WatkinsonLRSt JohnAPenberthyLA. Investigation into paediatric bilirubin analyses in Australia and New Zealand. J Clin Pathol1982; 35: 52–8.
24.
BlijenbergBGLeijnseB. A survey report on the determination of total bilirubin in neonatal samples. J Clin Chem Clin Biochem1980; 18: 27–30.
25.
BlijenbergBGRoeteringHALeijnseB. Reflections on the standardization of total bilirubin in neonatal serum. J Clin Chem Clin Biochem1987; 25: 177–81.
26.
KirwanNAAnnanW. A laboratory evaluation of three bilirubinometers. DHSS Scientific & Technical Branch, STB3A/A016, 1982.
27.
BijsterPVaderHLVinkCLJ. On the standardisation of the direct spectrophotometric bilirubin determination. Ann Clin Biochem1981; 18: 102–5.
28.
VinkKLJSchuurmanWvan GansewinkelR. Use of the caffeine reagent in direct spectrophotometry of bilirubin. Clin Chem1986; 32: 1389–93.
29.
VinkKLJSchuurmanWvan GansewinkelR. Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent. Clin Chem1988; 34: 67–70.
30.
FranziniCCattozzoG. Caffeine-splitting of bilirubin/albumin complex: Its relevance to the spectrophotometry of bilirubin in serum. Clin Chem1987; 33: 597–9.
31.
WuT-WDappenGMPowersDMLoDHRandRNSpaydRW. The Kodak Ektachem clinical chemistry slide for measurement of bilirubin in newborn: Principles and performance. Clin Chem1982; 28: 2366–72.
32.
YamanouchiIYanauchiYIgarashiI. Transculaneous bilirubinometry: Preliminary studies of noninvasive transcutaneous bilirubin meter in the Okayama National Hospital. Pediatrics1980; 65: 195–202.
33.
MaiselsMJConradS. Transcutaneous bilirubin measurements in full-term infants. Pediatrics1982; 70: 464–7.
34.
KennyMMetzgerGLandichoDChowJ. Transcutaneous bilirubin monitoring of newborns. Ann NY Acad Sri1984; 428: 251–62.
35.
Sheridan-PereiraMGormanW. Transcutaneous bilirubinometry: An evaluation. Arch Dis Childh1982; 57: 708–20.
36.
SchubiegerGMullisPKorberHR. Transcutaneous determination of bilirubin in the neonatal department: An analysis of its uses. Helv Paediatr Acta1986; 41: 183–6.
37.
BillingBHLatheGH. The excretion of bilirubin as an ester glucuronide giving the direct van den Bergh reaction. Biochem J1956; 63: 6P.
38.
DoumasBTKwok-CheungPPPerryBW. Candidate reference method for determination of total bilirubin in serum: Development and validation. Clin Chem1985; 31: 1779–89.
39.
ColePGLatheGHBillingBH. Separation of the bile pigments of serum, bile, and urine. Biochem J1954; 57: 514–18.
40.
DangerfieldWGFinlaysonR. Estimation of bilirubin in serum. J Clin Pathol1953; 6: 173–7.
41.
LandisJBPardueHL. Kinetics of the reactions of unconjugated and conjugated bilirubins with p-diazobenzenesulfonic acid. Clin Chem1978; 24: 1690–9.
42.
DoumasBTPerryBWSasseEATraumfjordJV. Standardisation in bilirubin assays: Evaluation of selected methods and stability of bilirubin solutions. Clin Chem1973; 19: 984–93.
43.
NovrosJSKochTRKnoblockEC. Improved method for accurate quantitation of total and conjugated bilirubin in serum. Clin Chem1979; 25: 1891–9.
44.
BillingBHaslamRWaldN. Bilirubin standards and the determination of bilirubin by manual and Technicon Auto Analyser methods. Ann Clin Biochem1971; 8: 21–30.
45.
ErtingshausenGByredFabiny DLTiffanyTOCaseySJ. Single-reagent method for rapid determination of total bilirubin with the ‘Centrifichem’ analyser. Clin Chem1973; 19: 1366–9.
46.
DappenGMSunbergMWWuT-WBabbBESchaefferJR. Diazo-based dry film for determination of total bilirubin in serum. Clin Chem1983; 29: 37–41.
47.
ThomasLPlischkeWStorzG. Evaluation of a quantitative solid-phase reagent system for determination of blood analytes. Experience with the analytes LDH, bilirubin, BUN, glucose and uric acid. Ann Clin Biochem1982; 19: 214–23.
48.
WestwoodA. Determination of total and direct bilirubin in plasma by means of a bichromatic method on a centrifugal analyser. Ann Clin Biochem1982; 19: 151–6.
49.
RyanFJMathiesJC. A total and direct bilirubin determination using an SDS procedure on the Abbot Bichromatic Analyser. Clin Biochem1983; 16: 254–8.
50.
MoriL. Modified Jendrassik-Grof method for bilirubins adapted to the Abbot Bichromatic Analyser. Clin Chem1978; 24: 1841–5.
SharmaAArtissJDStrandberghDRZakB. Enzymic clearing of lipaemic serum following total parenteral nutrition: Determination of neonatal bilirubin as a model. J Clin Chem Clin Biochem1986; 24: 571–5.
53.
PoonRHinbergIH. Indican interference with six commercial procedures for measuring total bilirubin. Clin Chem1985; 31: 92–4.
54.
SchullBCLeesHLiPK. Mechanism of interference by hemoglobin in the determination of total bilirubin. I. Method of Malloy-Evelyn. Clin Chem1980; 26: 22–5.
55.
SchullBCLeesHLiPK. Mechanism of interference by hemoglobin in the determination of total bilirubin. II. Method of Jendrassik-Grof. Clin Chem1980; 26: 26–9.
56.
Van der Woerd-De LangeJAGuderWGSchleicherEPaetzkeIschleithoffMWielandOH. Studies on interference by hemoglobin in the determination of bilirubin. J Clin Chem Clin Biochem1983; 21: 437–43.
57.
LoDHWuT-W. Assessment of the fundamental accuracy of the Jendrassik-Grof total and direct bilirubin assays. Clin Chem1983; 29: 31–6.
58.
PerryBDoumasBTBuffoneGGlickMOuC-NRyderK. Measurement of total bilirubin by use of bilirubin oxidase. Clin Chem1986; 32: 329–32.
59.
KosakaAYamamotoCMorishitaYNakaneK. Enzymatic determination of bilirubin fractions in serum. Clin Biochem1987; 20: 451–8.
60.
MullonCJ-PLangerR. Determination of conjugated and total bilirubin in serum of neonates, with use of bilirubin oxidase. Clin Chem1987; 33: 1822–5.
61.
OtsujiSMizunoKItoSKawaharaSKaiM. A new enzymatic approach for estimating total and direct bilirubin. Clin Biochem1988; 21: 33–8.
62.
CashoreWJOhWBlumbergWEEisingerJLamolaAA. Rapid fluorometric assay of bilirubin and bilirubin binding capacity in blood of jaundiced neonates: Comparsions with other methods. Pediatrics1980; 66: 411–16.
63.
KochTRAkingbeO. Feasibility of measuring free and total bilirubin electrochemically in serum. Clin Chem1981; 27: 1295–9.
64.
BlanckaertNKabraPMFarinaFAStaffordBEMartonLJSchmidR. Measurement of bilirubin and its monoconjugates and diconjugates in human serum by alkaline methanolysis and high performance liquid chromatography. J Lab Clin Med1980; 94: 198–212.
65.
WuT-WSullivanSS. ‘Biliprotein’ in adult icteric serum: Demonstrated by an extension of the alkaline methanolysis procedure. Clin Chem1982; 28: 2398–404.
66.
MuracaMBlanckaertN. Liquid-chromatographic assay and identification of mono- and di-ester conjugates of bilirubin in normal serum. Clin Chem1983; 29: 1767–71.
67.
LauffJJKasperMEAmbroseRT. Quantitative liquid-chromatographic estimation of bilirubin species in pathological serum. Clin Chem1983; 29: 800–5.
68.
AdachiYInufusaHYamashitaM. Clinical application of serum bilirubin fractionation by simplified liquid chromatography. Clin Chem1988; 34: 385–8.
69.
SeligsonDSeligsonHWuT-W. An anion-exchange chromatographic method for measuring bilirubin covalently bound to albumin. Clin Chem1985; 31: 1317–21.
70.
SundbergMWLauffJJWeissJSDappenGMWuT-WSpaydRWSeligsonD. Estimation of unconjugated, conjugated and ‘delta’ bilirubin fraction in serum by use of two coated thin films. Clin Chem1984; 30: 1314–17.
71.
ChanKMScottMGWuT-W. Inaccurate values for direct bilirubin with some commonly used direct bilirubin procedures. Clin Chem1985; 31: 1560–3.
72.
DoumasBTPerryBWJendrzejczakBKatonaV. Pitfalls in the American Monitor kit methods for determination of total and ‘direct’ bilirubin. Clin Chem1982; 28: 2305–8.
73.
DoumasBTPerryBJendrzejczakBDavisL. Measurement of direct bilirubin by use of bilirubin oxidase. Clin Chem1987; 33: 1349–53.
74.
AhlforsCE. Serious analytical and conceptual errors in bilirubin binding studies. Pediatrics1981; 67; 935–6.
75.
GoldfinchMEMaguireGA. Investigation of the use of bilirubin oxidase to measure the apparent unbound bilirubin concentration in human plasma. Ann Clin Biochem1988; 25: 73–7.
76.
JacobsenLWennbergRP. Determination of unbound bilirubin in the serum of newborns. Clin Chem1974; 20: 783–9.
77.
KapitulnikJBlondheimSHKaufmannNA. Sephadex absorption of bilirubin from neonatal and adult serum. Clin chem1972; 18: 43–7.
78.
LamolaAAEisingerJBlumbergWEPatelSCFloresJ. Fluorometric study of the partition of bilirubin among blood components: Basis for rapid microassays of bilirubin and bilirubin capacity in whole blood. Anal Biochem1979; 100: 25–42.
79.
LevineRL. Fluorescence-quenching studies of the binding of bilirubin to albumin. Clin Chem1977; 23: 2292–301.
80.
CashoreWJMoninPJPOhW. Serum bilirubin binding capacity and free bilirubin concentration: A comparison between Sephadex G-25 filtration and peroxidase oxidation techniques. Pediatr Res1978; 12: 195–8.
81.
TurnellDC. The calibration of total serum bilirubin assays. Ann Clin Biochem1985; 22: 217–21.
82.
DybkaerRHertzH. A reference for the determination of bilirubin concentration in serum using cyanide-formamide for enrichment of serum. Scand J Lab Clin Invest1970; 25: 151–60.
83.
BlijenbergBGRoeteringHAde VosALeijnseB. Further studies on the standardization of neonatal bilirubin. J Clin Chem Clin Biochem1987; 25: 737–41.
84.
VinkKLJvan DreumelRJSchuurmanWWikkelingHvan GansewinkelRPhielixCJKoedamHC. A candidate standard for use in calibration of total bilirubin in serum. Clin Chem1987; 33: 1817–21.
85.
St JohnAPenberthyLA. An inter-laboratory survey of paediatric bilirubin analyses. J Clin Pathol1979; 32: 794–7.
86.
AmentaJSSilvermanJA. Commercially prepared bilirubin standards may not be valid for use with direct spectrophotometric assay. Clin Chem1982; 28: 1812.
87.
RohleGSchlebuschHGeilenkeuserWJKruseR. External quality control in the determination of neonatal bilirubin. An approach to the improvement of results. J Clin Chem Clin Biochem1988; 26: 441–6.
88.
JohnsonKR. An external quality assessment scheme for total bilirubin. Ann Clin Biochem1988; 25: 78–84.
89.
Standards and Recommendations for Hospital Care of Newborn Infants, 6th EditionAmerican Academy of Pediatrics: 1977; 95.
90.
FraserCG. The application of theoretical goals based on biological variation data in proficiency testing. Arch Pathol Lab Med1988; 112: 404–15.
