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Task
Objectives
1. Explain formation, excretion, and clinical significance of direct, indirect and total bilirubin.
2. Perform a total bilirubin determination.
3. Perform a direct bilirubin determination.
Introduction
Like so many other substances measured in clinical chemistry laboratories, bilirubin is a waste
product. Bilirubin, the principle pigment in bile, is derived from the breakdown of hemoglobin.
After several degradation steps, the free bilirubin becomes bound by albumin and is transported
through the blood to the liver. This bilirubin is not soluble in water, and is referred to as insoluble,
indirect, or unconjugated. In the liver, bilirubin is rendered soluble by conjugation with
glucuronide. The water-soluble bilirubin, called direct or conjugated, is transported along with
other bile constituents into the bile ducts, then to the intestines. In the intestines, bacterial enzyme
action converts bilirubin to several related compounds, collectively referred to as urobilinogen.
Early methods for bilirubin estimation were based on measurement of its oxidation product,
biliverdin or on assessment of the icteric index. Introduction of the diazo reaction for bilirubin by
van den Bergh in 1918 led to its widespread adoption for quantitating the pigment in serum. Van
den Bergh and Muller found that bilirubin in normal serum reacted with Ehrlich's diazo reagent
(diazotized sulfanilic acid) when alcohol was added. Their observation that bile pigment reacted
with the diazo reagent without the addition of alcohol led to the recognition that some change in
bilirubin had been affected by the liver.
Bilirubin that reacts with the diazo reagent without the addition of alcohol is called “direct” or
conjugated while the form that reacts only in the presence of alcohol is called “indirect” or
unconjugated.
A low concentration of bilirubin is found in normal plasma, almost all of which is indirect. The sum
of the direct and indirect forms (or conjugated and unconjugated) is termed total bilirubin. Routine
analytical procedures exist for the determination of total bilirubin and for the measurement of
direct bilirubin. The indirect fraction is obtained by subtracting the direct value from the total
value. The determination of direct as well as total bilirubin is used in differentiating certain types
of jaundice.
Clinical Significance
Any increase in formation or retention of bilirubin by the body may result in jaundice, a condition
characterized by an increase in the bilirubin level in the serum and the presence of a yellowish
pigmentation in the skin.
organic or genetic causes. Post-hepatic jaundice refers to anatomic obstruction of the extra-
hepatic biliary tree. The most common causes of jaundice are liver disease and blockage of the
common bile duct. It is necessary to distinguish between the causes of jaundice early in the
disease prior to the onset of complications, as the course of treatment is dependent on the cause
of the jaundice.
Hemolytic jaundice is caused by overproduction of bilirubin due to excessive hemolysis and the
inability of the liver to adequately remove this pigment from the blood. This condition is usually
associated with elevated values of serum indirect bilirubin.
Cirrhosis of the liver and infectious or toxic hepatitis are caused by some type of intrahepatic
obstruction, where production of bilirubin is not increased, but accumulates and is discharged back
into the blood. In these conditions, the indirect form of bilirubin predominates in the early phase,
but as liver damage progresses the direct form also becomes elevated.
Obstructive jaundice, caused by a post-hepatic blockage of the larger bile passages, particularly
the common bile duct, results in a reflux of bilirubin into the blood. This condition, when
uncomplicated, is associated with elevated serum bilirubin only of the direct type.
Measurement of total bilirubin and determination of the direct and indirect fractions is important
in routine screening for and the differential diagnosis of jaundice.
Specimens for bilirubin determination should be protected from light, since bilirubin is light-
sensitive and will break down under exposure.
Methods of Determination
1. Van den Bergh, Malloy and Evelyn Reaction — In an aqueous solution, Ehrlich's diazo
reagent reacts with the direct bilirubin in the serum to form a pink to reddish-purple colored
compound (azobilirubin). It is read at one minute. In a 50% methyl alcohol solution, Ehrlich's
diazo reagent reacts with the total bilirubin in the serum to form a pink to reddish-purple
colored compound. (Read at 30 minutes.)
3. ASTRA — The ASTRA System Direct Bilirubin Chemistry Module employs a modification of
the Jendrassik-Grof rate method.
4. ACA
a. Conjugated Bilirubin – Conjugated bilirubin reacts with DSA under acid conditions to
form a red chromophore. The absorbance due to the chromophore is directly
proportional to the conjugated bilirubin in the sample and is measured using a two-filter
(540-600 nm) end point technique.
b. Total Bilirubin – Total bilirubin reacts with DSA under acid conditions to form a red
chromophore. Lithium dodecyl sulfate (LDS) is employed to solubilize the unconjugated
bilirubin. The absorbance due to the chromophore is directly proportional to the bilirubin
in the sample and is measured using a two-filter (540-600 nm) end point technique.
LDS
Bilirubin + DSA + H+ Red chromophore
(non-absorbing at 540 nm) (absorbs at 540 nm)
c. Neonatal bilirubin (up to 21 days) – The absorbance of the sample, measured using a
two-filter (452-540 nm) differential technique is directly proportional to the bilirubin
concentration. Absorbance at 452 nm is due to the bilirubin concentration, and, if
present, hemoglobin. At 540 nm, bilirubin does not absorb, while hemoglobin exhibits
the same absorbance as it does at 452 nm. The use of 540 nm as the blanking
wavelength thus eliminates any hemoglobin contribution from the total absorbance at
452 nm.
Procedure
Principle of Reaction
Bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. The color of this derivative
is pH dependent, occurring as pink in acid or neutral medium and blue under alkaline conditions.
Direct (conjugated) bilirubin couples with diazotized sulfanilic acid (p-diazobenzenesulfonic acid),
forming a blue color at alkaline pH.
Direct bilirubin (conjugated) + diazotized sulfanilic acid alkaline pH > blue color azobilirubin
2. alkaline tartrate – CAUTION: Strong base. Avoid contact with skin and clothing.
3. HCl (0.05 N)
4. Diazo Reagent (sulfanilic acid, sodium nitrite). Reconstitute one vial Diazo with 6.0 mL HCl.
Stable five days at 2-6°C.
5. Cysteine Reagent. Prepare by adding 10.5 mL DIH2 O. Cap, shake. Stable three months
(room temperature) in the dark.
6. Bilirubin reference. Assayed dry preparation containing bilirubin in a protein base for use as
a control or for calibration purposes. The actual bilirubin concentration appears on the vial
label.
Fresh serum is recommended, but heparinized plasma is also acceptable. Specimens must be
protected from both artificial light and sunlight during processing and storage as bilirubin will
undergo auto-oxidation to biliverdin.. The use of a serum blank eliminates interference from
hemolysis and lipemia.
1. Reconstitute bilirubin reference with 3.0 mL water. Let stand for several minutes and swirl
or invert to mix.
2. Number three test tubes and pipet solutions as indicated in the chart below
Bilirubin
(mg/dL) - (F)
Dilution x listed value
Multiplication of Bilirubin
Tube # Bilirubin Reference W ater Factor (F) Reference Absorbance
1 0.05 mL 0.15 mL 0.25
2 0.10 mL 0.10 mL 0.50
3 0.20 mL – 1.00
3. To each tube add in the sequence shown: (mix after each addition)
4. Transfer solutions to cuvets and record absorbance of all tubes using water as a reference
at 600 nm. (Read within 30 minutes.)
5. Calculate the bilirubin concentrations for each tube by multiplying the listed value for the
bilirubin reference by the appropriate dilution factor and record.
Procedural Notes
1. For screening purposes, the serum blank may be omitted, since the contribution by serum
to the final absorbance in this procedure is generally minor.
2. A serum blank should be included primarily when assaying highly turbid sera or control or
grossly hemolyzed specimens.
4. When the serum blank is omitted, the total and direct bilirubin tubes are read versus water
as a reference.
Working Procedure
3. Transfer to cuvets and read absorbance of all tubes, including blank using DI water as a
reference at 600 nm.
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. Record all results on worksheet.
Normal Values
Total Direct
Adults 0.2-1.2 mg/dL 0.3 mg/dL
Infants 1.0-12.0 mg/dL —
Name
Date
Bilirubin Worksheets
I. Calibration Curve
Wavelength _____________
Linearity _____________ Spectrophotometer Used _____________________
Control 1 ____________
Control 2 ____________
Control 1 ____________
Control 2 ____________
Control 1 ____________
Control 2 ____________
NOTES:
1. Show at least one example calculation for indirect bilirubin on the back of this page.
2. The bilirubin curve must accompany the results sheet.
3. See the back of this page to evaluate and report control results.
Name
Date
Study Questions
Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each
question is worth one point. Using lecture notes, reading assignments and information presented
in this lab, answer the following questions.
3. List three acceptable adjectives or names for the bilirubin molecule before it is conjugated.
(3 points)
5. During the conjugation process, bilirubin will be combined with what substance?
6. List three acceptable adjectives or names for the bilirubin molecule after it has been
conjugated. (3 points)
8. List reagents needed to perform the direct bilirubin procedure. (½ point each, 2 points total)
9. What different reagent(s) are needed for the total bilirubin procedure? What is (are) their
purpose(s)? (2 points)
11. What special procedures are needed in the handling of bilirubin samples? Why?
13. Associate the different basic types of jaundice with increased levels of bilirubin by completing
the following chart. (3 points)