unit: total and direct bilirubin

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UNIT: Total and Direct Bilirubin 13bili.wpd

Task

Determination of total and direct bilirubin.

Objectives

Upon completion of this exercise, the student will be able to:

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 wasteproduct. Bilirubin, the principle pigment in bile, is derived from the breakdown of hemoglobin.

After several degradation steps, thefree 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 asinsoluble,indirect, or unconjugated. In the liver, bilirubin is rendered soluble by conjugation with

glucuronide. The water-soluble bilirubin, calleddirect or conjugated, is transported along with

other bile constituents into the bile ducts, then to the intestines. In the intestines, bacterial enzymeaction 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 byvan den Bergh in 1918 led to its widespread adoption for quantitating the pigment in serum. Vanden 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 reactedwith 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 orconjugated while the form that reacts only in the presence of alcohol is called indirect orunconjugated.

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 termedtotalbilirubin. Routine

analytical procedures exist for the determination oftotal 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 conditioncharacterized by an increase in the bilirubin level in the serum and the presence of a yellowishpigmentation in the skin.

Jaundice may be classified as prehepatic, hepatic, or post-hepatic. Inprehepatic jaundice, excess

bilirubin production (hemolysis) is responsible. Hepatic jaundice occurs when either the removal

of bilirubin from the blood or conjugation of bilirubin by the liver is defective. This can have


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UNIT: Total and Direct Bilirubin (continued)

F 114 C MLAB 2401 - Clinical Chemistry Lab Manual

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 thecommon bile duct. It is necessary to distinguish between the causes of jaundice early in thedisease prior to the onset of complications, as the course of treatment is dependent on the causeof the jaundice.

Hemolytic jaundice is caused by overproduction of bilirubin due to excessive hemolysis and theinability of the liver to adequately remove this pigment from the blood. This condition is usuallyassociated with elevated values of serum indirectbilirubin.

Cirrhosis of the liver and infectious or toxic hepatitis are caused by some type of intrahepaticobstruction, where production of bilirubin is not increased, but accumulates and is discharged backinto the blood. In these conditions, theindirect 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, particularlythe common bile duct, results in a reflux of bilirubin into the blood. This condition, when

uncomplicated, is associated with elevated serum bilirubin only of thedirecttype.

Measurement of total bilirubin and determination of the direct and indirect fractions is importantin 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'sdiazo reagent reacts with the total bilirubin in the serum to form a pink to reddish-purplecolored compound. (Read at 30 minutes.)

2. Methods of Jendrassik and Grof Serum or plasma is added to a solution of sodium

acetate and caffeine-sodium benzoate. The sodium acetate buffers the pH of the diazoreaction, while the caffeine-sodium benzoate accelerates the coupling of bilirubin withdiazotized sulfanilic acid. The azobilirubin color develops within 10 minutes. (Anaccelerating agent facilitates the coupling of albumin-bound bilirubin with the diazo reagent.)

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 toform a red chromophore. The absorbance due to the chromophore is directlyproportional to the conjugated bilirubin in the sample and is measured using a two-filter(540-600 nm) end point technique.

Conjugated bilirubin + DSA + H 6 Red chromophore+

(non-absorbing at 540 nm) (absorbs at 540 nm)


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MLAB 2401 - Clinical Chemistry Lab Manual C F 115

b. Total Bilirubin Total bilirubin reacts with DSA under acid conditions to form a redchromophore. Lithium dodecyl sulfate (LDS) is employed to solubilize the unconjugatedbilirubin. The absorbance due to the chromophore is directly proportional to the bilirubinin the sample and is measured using a two-filter (540-600 nm) end point technique.

Bilirubin + DSA + H Red chromophore+ LDS

(non-absorbing at 540 nm) (absorbs at 540 nm)

c. Neonatal bilirubin (up to 21 days) The absorbance of the sample, measured using atwo-filter (452-540 nm) differential technique is directly proportional to the bilirubinconcentration. Absorbance at 452 nm is due to the bilirubin concentration, and, ifpresent, hemoglobin. At 540 nm, bilirubin does not absorb, while hemoglobin exhibitsthe same absorbance as it does at 452 nm. The use of 540 nm as the blankingwavelength thus eliminates any hemoglobin contribution from the total absorbance at452 nm.

Bilirubin in newborn babies can be read in this direct spectrophotometric procedure inpart due to the fact that the normal range is much higher than for adults. In addition,carotene and other dietary pigments prevent adult and specimens from older childrenfrom being suitable.

Procedure

Total and Direct Bilirubin (Sigma #605) Quantitative, Colorimetric

Principle of Reaction

Bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. The color of this derivativeis 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.

> blue color azobilirubinDirect bilirubin (conjugated) + diazotized sulfanilic acid alkalinepH

Indirect (unconjugated) bilirubin is diazotized only in the presence of an accelerating agent,

caffeine-benzoate-acetate mixture. Thus, the blue azobilirubin produced in mixtures containing

accelerating agent originates from both theDirect and Indirect fractions and reflects the Total

bilirubin concentration.

Total bilirubin + caffeine-benzoate-acetate mixture + diazotized sulfanilic acid6 azobilirubin

Supplies and Reagents

1. caffeine reagent (caffeine, sodium benzoate, sodium acetate)

2. alkaline tartrate CAUTION: Strong base. Avoid contact with skin and clothing.


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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 2-6C.

25. Cysteine Reagent. Prepare by adding 10.5 mL DIH 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 asa control or for calibration purposes. The actual bilirubin concentration appears on the viallabel.

7. Standard, controls (2), and unknowns.

Specimen Collection and Storage

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 willundergo auto-oxidation to biliverdin.. The use of a serum blank eliminates interference fromhemolysis and lipemia.

Preparation of Calibration Curve

1. Reconstitute bilirubin reference with 3.0 mL water. Let stand for several minutes and swirlor invert to mix.

2. Number three test tubes and pipet solutions as indicated in the chart below

T ube # Bilirub in Reference W ater

Dilution

Multiplication

Factor (F)

Bilirubin

(mg/dL) - (F)

x listed value

of B ilirubin

Refe rence A bsorbance

123

0.05 mL0.10 mL0.20 mL

0.15 mL0.10 mL

0.250.501.00

3. To each tube add in the sequence shown: (mix after each addition)

a. 1.0 mL caffeine reagentb. 0.5 mL diazo reagentc. 0.1 mL cysteine solutiond. 1.5 mL alkaline tartrate

4. Transfer solutions to cuvets and record absorbance of all tubes using water as a referenceat 600 nm. (Read within 30 minutes.)

5. Calculate the bilirubin concentrations for each tube by multiplying the listed value for thebilirubin reference by the appropriate dilution factor and record.


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6. Plot a calibration curve of the absorbance vs. concentration.

Procedural Notes

1. For screening purposes, the serum blank may be omitted, since the contribution by serumto the final absorbance in this procedure is generally minor.

2. A serum blank should be included primarily when assaying highly turbid sera or control orgrossly hemolyzed specimens.

3. Results are not significantly affected by hemoglobin concentrations up to 280 mg/dL.

4. When the serum blank is omitted, the total and direct bilirubin tubes are read versus wateras a reference.

Working Procedure

1. Set up Blank tube onlyon specimens that are hemolyzed or lipemic.

2. To appropriately labeled test tubes add the following:

Blank Total Directtube tube tube

a. serum 0.2 0.2 0.2b. hydrochloric acid 0.5 1.0c. caffeine reagent 1.0 1.0 d. diazo reagent 0.5 0.5

Mix welle. cysteine solution 0.1 0.1 0.1

Mix wellf. alkaline tartrate 1.5 1.5 1.5

Mix well

3. Transfer to cuvets and read absorbance of all tubes, including blank using DI water as areference 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 thedifference between the total and the direct. Record all results on worksheet.

Normal Values

Total DirectAdults 0.2-1.2 mg/dL 0.3 mg/dLInfants 1.0-12.0 mg/dL


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NameDate

Bilirubin Worksheets

I. Calibration Curve

Wavelength _____________Linearity _____________ Spectrophotometer Used _____________________

Identification Absorbance Reference Concentration Value *

Standard Tube 1 ____________



* see Preparation of Calibration Curve

II. Total Bilirubin

IdentificationBlanks

AbsorbanceTests

AbsorbanceCorrected

AbsorbanceConcentration (units)

Control 1 ____________

Control 2 ____________

Calculation formula(s) and examples

III. Direct Bilirubin

IdentificationBlanks

AbsorbanceTests

AbsorbanceCorrected

AbsorbanceConcentration (units)

Control 1 ____________

Control 2 ____________



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IV. Indirect Bilirubin

Identification Concentration (units)

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.

Total Bilirubin Quality Control

Your Results Controls range of expectedresults.

In control?Yes / No

Level 1 ID______________

Level 2ID_______________

Accepting Patient Results? Reason

Direct Bilirubin Quality Control

Your Results Controls range of expected

results.

In control?

Yes / No

Level 1 ID______________

Level 2ID_______________

Accepting Patient Results? Reason


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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 presentedin this lab, answer the following questions.

1. Describe the relationship between hemoglobin and bilirubin.

2. Free bilirubin appears in the plasma attached to .

3. List three acceptable adjectives or names for the bilirubin molecule before it is conjugated.(3 points)

4. The conjugation of bilirubin occurs at what location?

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 beenconjugated. (3 points)

7. How are bilirubin values used?

8. List reagents needed to perform the direct bilirubin procedure. ( point each, 2 points total)

9. What differentreagent(s) are needed for the total bilirubin procedure? What is (are) their

purpose(s)? (2 points)


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10. How is urobilinogen related to bilirubin?

11. What special procedures are needed in the handling of bilirubin samples? Why?

12. What components are in Diazo Reagent?

13. Associate the different basic types of jaundice with increased levels of bilirubin by completing

the following chart. (3 points)

increased bilirubin levels seen / often associated in this type of jaundice

indirect

direct

total

unit: total and direct bilirubin

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