1. Kristin Palladino, M.S., MLS(ASCP)CM Clinical Chemistry
Bilirubin
Kristin Palladino, M.S., MLS(ASCP)CM
Clinical Chemistry

2. Bilirubin The yellow breakdown product of normal heme catabolism
The principle pigment in bile
Bilirubin circulates in the bloodstream in two forms:
Indirect (Unconjugated) Bilirubin
Direct (Conjugated) Bilirubin

3. Indirect Bilirubin
Bound to albumin and transported through the blood to the liver
Not soluble in water (not excreted in urine)
Lipid soluble
Can pass the blood brain barrier when albumin binding is exceeded
Unconjugated hyperbilirubinaemia in a newborn leads to irreversible damage manifesting as seizures, abnormal reflexes and eye movements (Kernicterus)

4. Direct Bilirubin
Made in the liver when bilirubin is conjugated with glucuronic acid by the enzyme glucuronyltransferase
This makes the bilirubin water soluble (can be excreted in the urine)
Cannot cross the blood brain barrier
Transported into the bile ducts, then to the intestines
Intestinal bacterial enzymes deconjugate and metabolize the bilirubin into:
Urobilinogen
Urobilin
Stercobilin

5. Bilirubin Metabolism

6. Normal Values
The sum of the direct and indirect forms is termed total bilirubin
Adults: mg/dl
Conjugated: mg/dl
Unconjugated: mg/dl
Infants: Normal values depend on the age of the baby in hours and whether the baby was premature or full term

7. Clinical Significance
Any increase in formation or retention of bilirubin in the body may result in jaundice
Characterized by an increase in the bilirubin level in the serum and presence of a yellowish pigmentation in the skin and sclera (white) of the eyes
Jaundice is classified as follows:
Prehepatic
Hepatic
Post-hepatic

8. Clinical Significance
Prehepatic Jaundice
Excess bilirubin production (hemolysis)
Hemolytic jaundice is caused by the overproduction of bilirubin due to excessive hemolysis and the inability of the liver to adequately remove the pigment from the blood
Associated with elevated values of serum indirect bilirubin

9. Clinical Significance
Hepatic Jaundice
Occurs when either the removal of bilirubin from the blood or conjugation of bilirubin by the liver is defective
Conjugation Failure
Crigler-Najjar Syndrome (No conjugated bilirubin produced)
Bilirubin Transport Disturbances
Gilbert’s Syndrome (most common hereditary cause of increased bilirubin; result of reduced activity of glucuronyltransferase). Increase in Indirect Bilirubin
Dubin-Johnson Syndrome (conjugated bilirubin can not get out of the cells). Increase in Direct Bilirubin
Hepatocellular damage or necrosis
Cirrhosis of the liver and infectious or toxic hepatitis
Intrahepatic Obstruction
Edema

10. Clinical Significance
Post-hepatic Jaundice
Obstruction of the larger bile passages, particularly the common bile duct, by stones, neoplasms, spasms or strictures
Results in reflux of bilirubin into the blood
Associated with an elevated serum bilirubin only of the direct type

11. Types of Jaundice

12. Specimens Serum or plasma (Li Heparin) Bilirubin is light sensitive
Samples must be protected from both artificial light and sunlight
Direct sunlight may cause up to 50% decrease in bilirubin within 1 hr
Avoid hemolysis and lipemia
Hemolysis results in a slight decease in bilirubin levels
Stable if kept in the dark for up to week 1 refrigerated or 3 months if frozen

13. Methods of Detection
Routine analytical procedures exist for the determination of total bilirubin and for the measurement of direct bilirubin
Indirect bilirubin is calculated by subtracting the direct value from the total value

14. Methods of Determination
Jendrassik-Grof
Total Bilirubin:
Serum or plasma is added to a solution of sodium acetate and caffeine-sodium benzoate
Sodium acetate buffers the pH of the diazo reaction
Caffeine-sodium benzoate accelerates the coupling of bilirubin (indirect) with diazotized sulfanilic acid
The pink azobilirubin color forms develops within 10 minutes
Alkaline tartrate (pH 13) is added to convert pink azobilirubin to blue azobilirubin
Ascorbic acid or cysteine destroys excess diazo reagent and helps prevent fading of azobilirubin
The absorbance is read at 600 nm

15. Methods of Determination
Jendrassik-Grof
Direct Bilirubin:
Serum or plasma is added to a dilute acid and diazotized sulfanilic acid
No accelerating agent (caffeine) is required
Pink azobilirubin forms and the reaction is stopped at 1 minute by the addition of ascorbic acid
Alkaline tartrate (pH 13) is added to convert the pink azobilirubin to blue azobilirubin
The absorbance is read at 600 nm

16. Method of Determination
Direct Bilirubin
Direct Bilirubin (conjugated) + diazotized sulfanilic acid blue color azobilirubin
Total Bilirubin
Total Bilirubin + Caffeine-benzoate-acetate mixture + diazotized sulfanilic acid blue color azobilirubin
Alkaline pH
Alkaline pH

17. Methods of Determination
Spectrophotometric for neonatal bilirubin:
Up to 21 days
The absorbance of the sample is measured using a two-filter ( nm) differential technique
Absorbance at 455 nm is due to the bilirubin concentration and if present, hemoglobin
At 575 nm, bilirubin does not absorb but hemoglobin does
Subtract absorbance at 575 nm to correct for hemoglobin interference

18. Bilirubin Procedure Sigma Diagnostics, Total and Direct Bilirubin
Quantitative, Colorimetric determination of total and direct bilirubin in serum or plasma at 600 nm
Two Methods are included:
We are using Method B (need to construct a calibration curve)

19. Bilirubin Procedure Calibration Procedure for Method B:
Reconstitute bilirubin reference with 3 mL water. Let stand for several minutes and then swirl or invert to mix.
Label 3 test tubes and pipet solutions as indicated in columns 2 and 3 (Sigma procedure page 3)
To each tube add in the sequence shown (mix after each addition)
1 mL caffeine reagent
0.5 mL diazo reagent
0.1 mL cysteine solution
1.5 mL alkaline tartrate
4. Transfer solutions to cuvets. Read and record absorbance of all tubes vs. water as reference at 600 nm
5. Calculate the bilirubin concentrations for each tube by multiplying the listed value for the bilirubin reference by the appropriate dilution factor and record
6. Plot a calibration curve of the absorbance values vs. corresponding bilirubin concentration (mg/dL).

20. **Bilirubin Procedure
Calibration Procedure for Method B:
Reconstitute bilirubin reference with 3 mL water. Let stand for several minutes and then swirl or invert to mix. (This has been done. Value of Ref is 9.0 mg/dl)
Label 6 cuvets T #1, T#2, T#3, TNC, TAbC, T Pat and 3 cuvets DNC, DAbC and D Pat Make dilutions of the Ref using the chart on page 3 of Sigma Procedure
T# mL Ref and 0.15 mL water T# mL Ref and 0.10 mL water T# mL Ref and no water Add 0.20 mL of NC, AbC and Patient to the appropriate cuvets
Add 1.0 mL of HCl to each of the three Direct tubes Add 1.0 mL of Caffeine to each of the six Total cuvets
3. To each cuvet add in the sequence shown (mix after each addition)
0.5 mL diazo reagent
0.1 mL 4% Ascorbic Acid solution
1.5 mL alkaline tartrate
4. Transfer solutions to cuvets. Read and record absorbance of all tubes vs. water as reference at 600 nm
5. Calculate the bilirubin concentrations for each tube by multiplying the listed value for the bilirubin reference by the appropriate dilution factor and record
6. Plot a calibration curve of the absorbance values vs. corresponding bilirubin concentration (mg/dL).

21. Bilirubin Procedure Working Procedure
Label 3 test tubes for Total Bilirubin: NC, AC, Patient. Label 3 test tubes for Direct Bilirubin: NC, AC, Patient.
To the appropriately labeled tubes, add the reagents in the order they are listed on the Procedure Table (Sigma Procedure page 2).
Transfer solutions to cuvets. Read and record absorbance of all tubes vs. water reference at 600 nm.
Direct bilirubin readings should be taken immediately. Total bilirubin readings may be taken after 1 minute but should be completed within 30 minutes.

22. Bilirubin Procedure Working Procedure
Label 3 test tubes for Total Bilirubin: NC, AC, Patient. Label 3 test tubes for Direct Bilirubin: NC, AC, Patient.
To the appropriately labeled tubes, add the reagents in the order they are listed on the Procedure Table (Sigma Procedure page 2).
Transfer solutions to cuvets. Read and record absorbance of all tubes vs. water reference at 600 nm.
Direct bilirubin readings should be taken immediately. Total bilirubin readings may be taken after 1 minute but should be completed within 30 minutes.

23. Bilirubin Procedure Results
Use the prepared calibration curve to determine the concentration of your unknown samples.
Determine total and direct bilirubin levels from the curve
The indirect bilirubin is the difference between the total and the direct.

24. References
Clinical Chemistry Lab Manual. Unit: Total and Direct Bilirubin.