BILIRUBIN

GROUP 2
INTRO TO BILIRUBIN
• A highly pigmented yellow compound which is a degradation product of
hemoglobin.
• 250-350 mg
• 85% - comes from the heme released from senescent red blood cells
• Remainder - originates from various heme-containing proteins found in other tissues,
notably the liver and muscles.
• Shunt bilirubin comes from the sources other than senescent red cells
BILIRUBIN METABOLISM
FRACTIONS OF BILIRUBIN

B1 B2

Bilirubin monoglucuronide Bilirubin diglucuronide

Unconjugated bilirubin Conjugated bilirubin

OTHER NAMES Water-insoluble bilirubin Water soluble bilirubin

Indirect bilirubin Direct bilirubin

Nonpolar bilirubin Polar bilirubin

Delta bilirubin is a bilirubin covalently bound with albumin, which is nontoxic and excreted
neither in urine nor in bile.
CLINICAL SIGNIFICANCE
OF BILIRUBIN
BILIRUBIN (CLINICAL SIGNIFICANCE)

• Major heme waste product

• Liver is the only organ that has the capacity to rid the body of heme
waste products
• Principal pigment in bile
• Breakdown of red blood cells
Unconjugated Bilirubin Conjugated Bilirubin
• B1 • Bilirubin diglucoronide
• Water-insoluble • Water soluble
• Indirect • Direct
• Nonpolar • Polar
 RBC

After 120 days: Oxidized to biliverdin Bile canaliculi

Phagocytized

Bilirubin Small intestine

Hemoglobin Heme

Transported to liver Mesobilirubinogen

Iron Globin

Sinusoidal spaces Urobilinogen

Liver or
Degraded to
bone
constituent
marrow for Endoplasmic reticulum Liver Urobilin Stercobilin
amino acids
reuse

Urine Feces
Conjugation of bilirubin
Reused
by body
• Hepatitis and cirrhosis are common conditions
that produce liver damage = bilirubinuria
• Detection of bilirubin can provide an early
indication of liver disease and can help
determine the cause of clinical jaundice
• Jaundice due to increased destruction of red
blood cells does not produce bilirubinuria
because serum bilirubin is in unconjugated
form which kidneys cannot excrete
JAUNDICE
• Characterized by yellow discoloration of skin and sclera
• Icterus
• Pre-hepatic, Hepatic, Post-hepatic

Pre-hepatic Hepatic Post-hepatic

• Acute hemolytic • Liver • Results from biliary
anemia • Result from impaired obstructive jaundice
• All conditions before cellular uptake, • Physical obstructions
the liver defective conjugation, (gallstones or tumors)
• Increased Total abnormal secretion of that prevent flow of
Bilirubin bilirubin by liver cell conjugated bilirubin
• Increased unconjugated into bile canaliculi
bilirubin • Increased total,
unconjugated and
conjugated bilirubin
HEPATIC JAUNDICE
Gilbert Syndrome
Crigler-Najjar Syndrome
Rotor’s Syndrome
Dubin-Johnson Syndrome
Physiologic Jaundice of the Newborn
Increased:Total, B1
Increased: Total, B1 ; Decreased: B2
Increased: Total, B2
Increased: Total, B2
Increased: Total, B1
REAGENT STRIP
PRINCIPLE FOR
BILIRUBIN
• Multistix Reagent Strip: If Bilirubin is present, color changes from cream
buff to tan. This uses the diazonium salt – diazotized 2,4-dichloroaniline.
• Detects 0.8mg/dL of Bilirubin
• Chemstrip reagent: Contains 6-dichlorobenzene diazonium
tetrafluoroborate as salt which changes color from pink to violet.
• Detects 0.5mg/dL of Bilirubin
 Acid medium
Bilirubin glucuronide + Diazonium salt Azodye
HOW IS IT REPORTED?
Qualitative Results Semi-Quantitative
Negative Results
Small Negative
Moderate 1+
Large 2+
3+
• Questionable Results should be confirmed through
IctoTest.
REACTION
INTERFERENCES OF
BILIRUBIN
FALSE POSITIVE
INTERFERENCES
FALSE POSTIVE INTERFERENCES
ARE CAUSED BY URINE PIGMENTS
PHENAZOPYRIDINE
(HIGHLY PIGMENTED
URINES)
INDICAN
(INTESTINAL
DISORDER)
METABOLITES OF
LODINE
FALSE NEGATIVE
INTERFERENCES
FALSE NEGATIVE INTERFERENCES
ARE CAUSED BY UNIMMEDIATE
TESTING OF URINE SPECIMEN
LIGHT EXPOSURE
ASCORBIC
ACID
HIGH NITRITE
CONCENTRATIONS
BIOCHEMICAL TEST:
ICTOTEST
ICTOTEST
ICTOTEST
• The test is easy to perform and gives timely and
accurate confirmatory urine bilirubin results in one
minute.

• Level of detection ≥ 0.05 to 0.1 mg bilirubin/dL in

urine (0.9–1.7 µmol/L).
ICTOTEST
CHEMICAL PRINCIPLES OF
THE PROCEDURE:
The reaction is based on the
coupling of a unique solid diazonium
salt with bilirubin in an acid medium
to give the blue or purple reaction.
ICTOTEST
p-nitrobenzenediazonium-
p-toluenesulfonate
Sulfosalicylic acid (SSA)
Sodium carbonate
Boric acid
5 SECONDS LATER
60 SECONDS LATER
RESULTS
Positive Negative
BILIRUBIN
PHYSICAL WITH OTHER
CHEMICAL PARAMETERS
AND MICROSCOPIC
CORRELATION OF
BILIRUBIN
COLOR

• Dark in colour, usually brown with yellow foam or

dark amber
 Bilirubin is NOT normally present in urine
 If the liver's function is impaired or when biliary drainage is
blocked, some of the conjugated bilirubin leaks out of the
hepatocytes and appears in the urine, turning it dark amber.
UROBILINOGEN

The result of bilirubin can be compared with the urobilinogen result.

• A negative test for bilirubin but positive for urobilinogen can indicate haemolytic
disease.
• A low or negative result for urobilinogen in a patient with a positive bilirubin test can
indicate a biliary/hepatic obstruction.
MICROSCOPIC EXAMINATION
Bilirubin Crystal
MICROSCOPIC EXAMINATION

Bilirubin Crystals

• Appearance
• Clumped granules or needles
• Orange-yellow color
• Viral hepatitis with tubular
damage

• Solubility
• Dissolve in acetic acid, HCl,
Acetone
EPITHELIAL CELL CASTS

• Casts containing RTE cells represent

the presence of advanced tubular
destruction

• Fragments of epithelial tissue may

also be attached to the cast matrix.
Bilirubin-stained RTE cells are seen in
cases of hepatitis.
FOAM TEST

• If the urine is a yellowish-brown or greenish-yellow color and bilirubin is suspected

• POSITIVE:YELLOW OR GREENISH-YELLOW FOAM IN URINE DEVELOPS

• FALSE-POSITIVE: WHEN URINE CONTAINS PHENAZOPYRIDINE