BILIRUBIN DETERMINATION

BILIRUBIN Evelyn Malloy Method

• About 200-300mg of bilirubin is produced daily in • A quantitative colorimetric diazo coupling method
healthy adults • It utilized a direct reading photoelectric colorimeter
• Composition: Total bilirubin, B1 and B2 PRINCIPLE:
• Van den Bergh Reaction (Diazotization)
• it is used as a marker to detect pre-hepatic, - Bilirubin reacts with diazotized sulfanilic acid to
hepatic and post-hepatic jaundice form pink to purple Azobilirubin
• An end product of hemoglobin metabolism
• Principal pigment in bile
• Used to assess function of the liver
• Jaundice - exceeds 2 mg/dL or up to 3 to 5mg/dL
MATERIALS:
• Visible icteria is present with >25 mg/L of bilirubin • Bilirubin standard total (4.0 mg/dL)
• Bilirubin standard solution is usually made up of • Bilirubin standard direct (2.0 mg/dL)
B1 • Bilirubin reagent (sulfanilic acid)
• Conjugated bilirubin (B2) - fraction that produce a • Sodium nitrite reagent (accelerator)
color in aqeous solution • Serum or plasma
• Unconjugated Bilirubin - fraction that produce a • Distilled water
color only after the addition of alcohol • Methanol (95%)
Purpose • Test tubes
• It measures the bilirubin in the blood. • Cuvets
• It is used to aid in the diagnosis of medical • Pipettes
• conditions such as jaundice, anemia, and liver • Spectrophotometer
disease. PROCEDUR
1. Pipet 100 ul of sample into a test tube.
Application 2. Add 1.4 ml of bilirubin reagent and one drop of
• Bilirubin levels in infants are measured to rule out bilirubin accelerator.
neonatal jaundice. 3. Mix well and stand at room temperature.
• Check to see whether your bile ducts, either in the 4. Read the absorbance at 550 nm.
liver or the gallbladder, are blocked. 5. Calculate the result against the values of the
• Aid in the detection of liver disease, particularly direct bilirubin standard. This is the first reading
hepatitis, or in the monitoring of its course. and corresponds to the value for direct bilirubin.
6. To the same mixture add 1. 5 ml of methanol.
7. Mix well and stand at room temperature
Specimen Collection & Handling
protected from direct light for exactly 5 minutes.
• Serum or plasma 8. Read the absorbance at 550 nm.
• No hemolysis 9. Calculate the result against the values of the
• Fasting Sample is preferred total bilirubin standard
• Stored in an amber bottle FORMULA:
• Specimens should be protected from bright light 1st Reading:
as bilirubin is photolabile

Methodology
• Evelyn Malloy Method 2nd Reading:
• Jendrassik and Grof Method
• Icterus Index
• Direct Spectrophotometry

 INTERPRETATION PROCEDURE:
Conversion factor:
• 17.1 in umol/L
Normal values:
• Direct Bilirubin - 0-0.35 mg/dL (0-5.985 umol/L
• Total Bilirubin - 0- 1.50 mg/dL (0-25.65 umol/L)
Increased:
• liver problem or disease, Viral hepatitis, A
reaction to drugs, Alcoholic liver disease,
Gallstones, Anemia, Cirrhosis, A reaction to
a blood transfusion, Gilbert syndrome

Jendrassik and Grof Method

• uses a timed-endpoint Diazo method to measure FORMULA:
the concentration of total bilirubin in serum or
plasma.

• Preferred method
• Candidate for reference method
• Specific for bilirubin
• More stable and sensitive end color
PRINCIPLE:
• Van den Bergh Reaction (Diazotization) The factor has to be checked by a calibration serum
- Direct conjugated bilirubin reacts with and adapted if necessary!
diazotized sulfanilic acid to form blue INTERPRETATION
Azobilirubin Conversion factor:
• 17.1 in umol/L
Normal values:
• Conjugated Bilirubin: 0-0.2 mg/dL (0-3 umol/L)
• Unconjugated Bilirubin: 0.2-0.8 mg/dL (3-14
umol/L
MATERIALS: • Total Bilirubin: 0.2-1.0 mg/dL (3-17 umol/L)
• 0.8% Ascorbic acid solution Increased:
• Caffeine benzoate acetate • liver problem or disease, Viral hepatitis, A
• Sulfanilic acid reaction to drugs, Alcoholic liver disease,
• Sodium nitrite Gallstones, Anemia, Cirrhosis, A reaction to
• Diazo reagent a blood transfusion, Gilbert syndrome
• Alkaline reagent Decreased:
• Distilled water • Independent predictors of advanced
• Test tubes inflammation and fibrosis in patients with
• Cuvets steatohepatitis, indicating the lack of antioxidant
• Pipettes
• Spectrophotometer

 SUMMARY:

Icterus Index
• Semi-quantitative method
• Add a standard solution of 0.1% potassium
dichromate

Direct Spectrophotometry
• For newborn
• 455 nm

Quality Control
• If more frequent control monitoring is required,
follow the established quality control procedures
for your laboratory.
• If quality control results do not meet acceptance
criteria defined by the laboratory, patient values
may be suspect. Follow the established quality
control procedures for the laboratory. Recalibration
may be necessary.
• Review quality control results and acceptance
criteria following a change of reagent or calibrator
lot.