BILIRUBIN DIAGNOSTIC SIGNIFICANCE

 Clinically hyperbilirubinemia appears as jaundice or

 Yellow breakdown product of normal heme
catabolism
 Excreted in the bile and urine (elevated levels may
indicate certain diseases)
 Responsible for the yellow color of bruises, the
background straw-yellow color of urine
 The brown color of feces (via its conversion to
stercobilin), and yellow discoloration in jaundice
 End product of hemoglobin and serve as a diagnostic
marker of liver and blood disorders
icterus
 Jaundice can be usually detected when the serum
bilirubin level exceeds 2.0 - 2.5 mg/dL
 When the level of bilirubin is between 1-2 mg/dL, it
is known as Latent Jaundice
 In neonates unconjugated bilirubin can cross BBB
(blood brain barrier) and thus lead to accumulation in
brain of neonates. This interfere with nervous system
development and permanent nervous impairment

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