BILIRUBIN DIAGNOSTIC SIGNIFICANCE
Clinically hyperbilirubinemia appears as jaundice or
Yellow breakdown product of normal heme icterus
catabolism Jaundice can be usually detected when the serum
Excreted in the bile and urine (elevated levels may bilirubin level exceeds 2.0 - 2.5 mg/dL
indicate certain diseases) When the level of bilirubin is between 1-2 mg/dL, it
Responsible for the yellow color of bruises, the is known as Latent Jaundice
background straw-yellow color of urine In neonates unconjugated bilirubin can cross BBB
The brown color of feces (via its conversion to (blood brain barrier) and thus lead to accumulation in
stercobilin), and yellow discoloration in jaundice brain of neonates. This interfere with nervous system
End product of hemoglobin and serve as a diagnostic development and permanent nervous impairment
marker of liver and blood disorders
FUNCTIONS DIFFERENCE BETWEEN CONJUGATED AND
Levels of serum bilirubin are inversely related to risk UNCONJUGATED BILIRUBIN
of certain heart disease CONJUGATED UNCONJUGATED
Present normally in bile Present normally in plasma
Acts as uncoupler in neonates and thus maintain body
Conjugated to glucoronic acid Conjugated to albumin
heat
Can be filtered by the kidney Cannot be filtered by the
Bile pigments such as biliverdin naturally possess kidney
significant anti-mutagenic and antioxidant properties Cannot cross BBB Can cross BBB
Biliverdin and bilirubin have shown to be potent
scavengers of peroxyl radicals
Inhibit the effcts of polycyclic aromatic hydocarbons, IN THE LABORATORY, BLIRUBIN IS MEASURED AS:
heterocylci amines, and antioxidants – all of which are 1. Total bilirubin
mutagens 2. Indirect or Unconjugated bilirubin
3. Direct or conjugated bilirubin
FORMS OF BILIRUBIN
Unconjugated Conjugated
Structure Free bilirubin; B1 Bilirubin CHANGES IN CONCENTRATION OF BILIRUBIN
diglucoronide; B2 IN THOSE WITH JAUNDICE
Other name Hemobilirubin; Choleglobin; TOTAL CONJUGATED
Hemolytic bilirubin; Cholebilirubin; One- TYPE OF JAUNDICE BILIRUBIN BILIRUBIN UNCONJUGATED
Prehepatic bilirubin minute bilirubin; PRE HEPATIC
Prompt bilirubin; HDN Normal
Cholestatic bilirubin; Physiologic Normal
Post-hepatic Diseases of the
Types of Non-polar Polar Newborn
Compound Ineffective Increase Normal Increase
Solubility Hematopoesis
Water Insoluble Soluble Crigler-Najar Decrease
Alcohol Soluble Soluble Syndrome
Van Den High affinity; Indirect Low affinity; Direct Gilberts Normal
Berg Syndrome
reaction HEPATIC
Urine: Absent Present Viral Increase Increase
Increase
Hemolytic Dubin Johnson Normal
Urine: Rotor syndorme Normal
Obstructive POST HEPATIC
Positive (+) Negative (–) Increase Increase Increase
Affinity to OBSTRUCTION
brain tissue
FRACTIONS OF BILIRUBIN Indirect Blilirubin = Total Bilirubin – Direct Bilirubin
Alpha – unconjugated
Beta – mono conjugated
Gamma – diconjugated
Delta – irreversibly bound to albumin. Usually seen
in solutions with problems in the extinction of
conjugated bilirubin
Note: Alpha beta and gamma have a directly out with diazo
reagent
DIFFERENTIAL DIAGNOSIS BILIRUBIN DETERMINATION
DIRECT SPECTRAL METHOD
DECREASED ICTERUS INDEX OF MUELENGRACHT
TRANSPORT - It is based on the degree of icterus in the serum or
INCREASED OR IMPAIRED BILIARY
PRODUCTION DECREASED EXCRETION OBSTURCTION plasma. As compared to the standard potassium
CONJUGATION
dichromate solution normal value is 2-6 icterus
Increased UB Increased CB
index units.
Hemolysis Gilbert’s Rotor’s CB/CBH
Syndrome Disease Syndrome
- Not reliable – pigments (carotene; xanthophyll)
Transfusion Crigler Najjar Dubin- Stricture JACKSON DIRECT BILIRUBINOMETER
Johnson - The serum is diluted with 15 m phosphate buffer
Transfusion Neonatal Cancer Tumor/ and the absorbance is read at 450 mm and 575 nm
Reaction Cancer using a narrow band filter. Standard methyl
Sepsis Cirrhosis Cirrhosis Pancreatitis orange at pH of 7.4
Burns Hepatitis Hepatitis Primary Note: it is very rapid and usually use among
Sclerotizing infants but not in adults due to interferences
Cholangitis - Bilirubin (mg/dl) = A450 - A575 x 50
Hemoglobino Drug Amyloidosis Parasitic
pathies Inhibition Infections
DIAZOTIZATION PRINCIPLE by Van den Berg Reaction
Pregnancy
– based on the reaction of bilirubin with diazotized sulfanilic
acid producing a colored pigment azobilirubin (red color)
FUNCTION PRE POST Evelyn-Malloy method
HEPATIC
TEST HEPATIC HEPATIC Jendrassik-Grof method
Total Bilirubin Normal/ Increased Increased Rand and Di Pasqua method
Increase Michaelson
Conjugated Normal Increased Increased
Gindler-Ishizaki
Unconjugated Normal/ Increased Normal
Anino Watson and Ducci
Increase
Stoner and Wisberg
Urobilinogen Normal/ Increased Decreased/
Thamhausser Anderson
Increase Negative
Fluorometry
Urine Color Dark Dark Dark
(Urobilinogen) (Urobilinogen (Conjugated Continuous Flow Technique by Gambino
+ Conjugated Bilirubin)
Bilirubin) Basis Evelyn- Jendrassik- Rand & Di
Stool Color Normal Normal Pale Malloy Grof Pasqua
Alkaline Product Red Blue Red
Phosphatase Normal Increased Increased Measured Violet Azobilirubin Azobilirubin
Level Azobilirubin
Alanine Incubation Db: 1 minute Db: 5 minutes 5 minutes
Transferase Period Tb: 15 minutes Tb: 10 minutes
and Aspartate Normal Increased Increased pH Acidic to Basic
Transferase Neutral
Levels Accelerator 50% Methanol Caffeine
Conjugated Sodium
Bilirubin in Absent Present Present Benzoate
Urine (Coupling
Promoter)
Buffer None Sodium
SPECIMEN CONSDERATION Acetate
Other Diazo Reagent Diazo Reagent Dicholor
Light sensitive analyte
Reagent and Blank; Benzene
Cover with foil or carbon paper both collection tubes Ascorbic Acid Diazonoum
and plasma or serum container after separation of use and Alkaline Chloride
of specialized tube which is amber colored Tartarate
Prevent life policies and lipemia
Diazo Reagent: 1.0% sulfanilic acid + 0.5% sodium nitrite
SERUM BILIRUBIN Diazo Blank: 1.5% HCL
Hyperbilirubinemia – plasma bilirubin exceeds 1 Ascorbic Acid – used to determine diazo reaction
mg/dL Alkaline Tartrate – shift peak absorbance of blilirubin from
586 to 600 nm (converts pink to blue)
Latent jaundice – between 1 to 2mg/dL
>2m g/dL – yellowish discoloration sclera Advantages of Jendrassik-Groff
conjunctiva skin mucous membrane resulting in Insensitive to pH change end 50 fold variation in change of
jaundice the concentration of the sample
Jaundice or Icterus – a condition characterized by Adequate optical sensitivity (low bilirubin); minimal
yellowish discoloration of the skin sclera and turbidity and relatively constant blank
mucous membrane Not affected by hemoglobin up to 750 mg/dL