UNIT: Total and Direct Bilirubin

Task Determination of total and direct bilirubin. Objectives Upon completion of this exercise, the student will be able to: 1. 2. 3.

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Explain formation, excretion, and clinical significance of direct, indirect and total bilirubin. Perform a total bilirubin determination. 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. Jaundice may be classified as prehepatic, hepatic, or post-hepatic. In prehepatic 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
MLAB 2401 - Clinical Chemistry Lab Manual C F 113

The most common causes of jaundice are liver disease and blockage of the common bile duct. Obstructive jaundice. 3. It is necessary to distinguish between the causes of jaundice early in the disease prior to the onset of complications. particularly the common bile duct. In these conditions. Ehrlich's diazo reagent reacts with the direct bilirubin in the serum to form a pink to reddish-purple colored compound (azobilirubin). 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. It is read at one minute. Van den Bergh. since bilirubin is lightsensitive and will break down under exposure. (An accelerating agent facilitates the coupling of albumin-bound bilirubin with the diazo reagent. the indirect form of bilirubin predominates in the early phase. In a 50% methyl alcohol solution.UNIT: Total and Direct Bilirubin (continued) organic or genetic causes. .) ASTRA — The ASTRA System Direct Bilirubin Chemistry Module employs a modification of the Jendrassik-Grof rate method.) Methods of Jendrassik and Grof — Serum or plasma is added to a solution of sodium acetate and caffeine-sodium benzoate. is associated with elevated serum bilirubin only of the direct type. results in a reflux of bilirubin into the blood. as the course of treatment is dependent on the cause of the jaundice. The sodium acetate buffers the pH of the diazo reaction. Cirrhosis of the liver and infectious or toxic hepatitis are caused by some type of intrahepatic obstruction. This condition is usually associated with elevated values of serum indirect bilirubin. This condition. The azobilirubin color develops within 10 minutes. but as liver damage progresses the direct form also becomes elevated. where production of bilirubin is not increased. ACA a. Malloy and Evelyn Reaction — In an aqueous solution. 4. when uncomplicated. while the caffeine-sodium benzoate accelerates the coupling of bilirubin with diazotized sulfanilic acid. (Read at 30 minutes. but accumulates and is discharged back into the blood. caused by a post-hepatic blockage of the larger bile passages. Conjugated Bilirubin – Conjugated bilirubin reacts with DSA under acid conditions to form a red chromophore. Methods of Determination 1. Conjugated bilirubin + DSA + H+ 6 Red chromophore (non-absorbing at 540 nm) (absorbs at 540 nm) F 114 C MLAB 2401 .Clinical Chemistry Lab Manual 2. Post-hepatic jaundice refers to anatomic obstruction of the extrahepatic biliary tree. Specimens for bilirubin determination should be protected from light. 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. Measurement of total bilirubin and determination of the direct and indirect fractions is important in routine screening for and the differential diagnosis of jaundice. Ehrlich's diazo reagent reacts with the total bilirubin in the serum to form a pink to reddish-purple colored compound.

UNIT: Total and Direct Bilirubin (continued) b. bilirubin does not absorb. sodium benzoate. carotene and other dietary pigments prevent adult and specimens from older children from being suitable. if present. Lithium dodecyl sulfate (LDS) is employed to solubilize the unconjugated bilirubin. Absorbance at 452 nm is due to the bilirubin concentration. Direct bilirubin (conjugated) + diazotized sulfanilic acid alkaline pH > blue color azobilirubin Indirect (unconjugated) bilirubin is diazotized only in the presence of an “accelerating” agent. Bilirubin in newborn babies can be read in this direct spectrophotometric procedure in part due to the fact that the normal range is much higher than for adults. Total Bilirubin – Total bilirubin reacts with DSA under acid conditions to form a red chromophore. LDS Bilirubin + DSA + H+ (non-absorbing at 540 nm) Red chromophore (absorbs at 540 nm) c. caffeine reagent (caffeine. Total bilirubin + caffeine-benzoate-acetate mixture + diazotized sulfanilic acid 6 azobilirubin Supplies and Reagents 1. In addition. measured using a two-filter (452-540 nm) differential technique is directly proportional to the bilirubin concentration. At 540 nm. occurring as pink in acid or neutral medium and blue under alkaline conditions. 2. The use of 540 nm as the blanking wavelength thus eliminates any hemoglobin contribution from the total absorbance at 452 nm. caffeine-benzoate-acetate mixture. while hemoglobin exhibits the same absorbance as it does at 452 nm. MLAB 2401 . Colorimetric Principle of Reaction Bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. and. forming a blue color at alkaline pH. Neonatal bilirubin (up to 21 days) – The absorbance of the sample. Procedure Total and Direct Bilirubin (Sigma #605) Quantitative. The color of this derivative is pH dependent.Clinical Chemistry Lab Manual C F 115 . Direct (conjugated) bilirubin couples with diazotized sulfanilic acid (p-diazobenzenesulfonic acid). 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. Thus. the blue azobilirubin produced in mixtures containing “accelerating” agent originates from both the Direct and Indirect fractions and reflects the Total bilirubin concentration. Avoid contact with skin and clothing. sodium acetate) alkaline tartrate – CAUTION: Strong base. hemoglobin.

Stable three months (room temperature) in the dark. Stable five days at 2-6°C. Number three test tubes and pipet solutions as indicated in the chart below Bilirubin (mg/dL) . 1.05 mL 0.25 0. The use of a serum blank eliminates interference from hemolysis and lipemia. (Read within 30 minutes..) Calculate the bilirubin concentrations for each tube by multiplying the listed value for the bilirubin reference by the appropriate dilution factor and record. and unknowns. d. Reconstitute one vial Diazo with 6.0 mL water.Clinical Chemistry Lab Manual .1 mL cysteine solution 1.20 mL 0.15 mL 0. Reconstitute bilirubin reference with 3. 7. but heparinized plasma is also acceptable.10 mL 0.00 3. Preparation of Calibration Curve 1. Transfer solutions to cuvets and record absorbance of all tubes using water as a reference at 600 nm. Cysteine Reagent.5 mL diazo reagent 0. 4. Cap.(F) x listed value of Bilirubin Reference Absorbance 2. c.0 mL HCl.UNIT: Total and Direct Bilirubin (continued) 3. The actual bilirubin concentration appears on the vial label. Specimen Collection and Storage Fresh serum is recommended. 6. 5. b. F 116 C MLAB 2401 .5 mL alkaline tartrate 4. HCl (0.50 1.5 mL DIH2 O. Standard. sodium nitrite). To each tube add in the sequence shown: (mix after each addition) a.05 N) Diazo Reagent (sulfanilic acid. Bilirubin reference. shake. controls (2). 5. Specimens must be protected from both artificial light and sunlight during processing and storage as bilirubin will undergo auto-oxidation to biliverdin. Tube # Bilirubin Reference W ater Dilution Multiplication Factor (F) 1 2 3 0.10 mL – 0.0 mL caffeine reagent 0. Assayed dry preparation containing bilirubin in a protein base for use as a control or for calibration purposes. Let stand for several minutes and swirl or invert to mix. Prepare by adding 10.

Clinical Chemistry Lab Manual C F 117 . e.0 — 0. Transfer to cuvets and read absorbance of all tubes.2-1.2 mg/dL 1. Normal Values Total 0. To appropriately labeled test tubes add the following: Blank tube 0. The indirect bilirubin is the difference between the total and the direct. 3.2 — 1. RESULTS Use the prepared calibration curve to determine the concentration of your unknown samples. serum hydrochloric acid caffeine reagent diazo reagent Mix well cysteine solution Mix well alkaline tartrate Mix well 3.1 1. For screening purposes.5 0. Record all results on worksheet.2 0. Procedural Notes 1.2 1.0 mg/dL Direct 0.5 a. Working Procedure 1.3 mg/dL — Adults Infants MLAB 2401 . 4. concentration. Results are not significantly affected by hemoglobin concentrations up to 280 mg/dL. since the contribution by serum to the final absorbance in this procedure is generally minor.0 0. c. the total and direct bilirubin tubes are read versus water as a reference.5 0.0 — 0.5 Direct tube 0. Set up Blank tube only on specimens that are hemolyzed or lipemic. Plot a calibration curve of the absorbance vs. d. f.0-12. 2.5 1. A serum blank should be included primarily when assaying highly turbid sera or control or grossly hemolyzed specimens. Determine total and direct bilirubin levels from the curve. 2. When the serum blank is omitted.5 Total tube 0. including blank using DI water as a reference at 600 nm. b.1 1. the serum blank may be omitted.UNIT: Total and Direct Bilirubin (continued) 6.1 1.

Clinical Chemistry Lab Manual .UNIT: Total and Direct Bilirubin (continued) F 118 C MLAB 2401 .

Total Bilirubin Identification Blank’s Absorbance Test’s Absorbance Corrected Absorbance Concentration (units) Control 1 ____________ Control 2 ____________ Calculation formula(s) and examples III. Calibration Curve Wavelength _____________ Linearity _____________ Identification Spectrophotometer Used _____________________ Absorbance Reference Concentration Value * Standard Tube 1 ____________ Standard Tube 2 ____________ Standard Tube 3 ____________ * see Preparation of Calibration Curve II.UNIT: Total and Direct Bilirubin (continued) Name Date Bilirubin Worksheets I. Direct Bilirubin Identification Blank’s Absorbance Test’s Absorbance Corrected Absorbance Concentration (units) Control 1 ____________ Control 2 ____________ Calculation formula(s) and examples MLAB 2401 .Clinical Chemistry Lab Manual C F 119 .

In control? Yes / No Direct Bilirubin Quality Control Your Results Level 1 ID______________ Level 2ID_______________ Accepting Patient Results? Reason Controls’ range of expected results. See the back of this page to evaluate and report control results. Total Bilirubin Quality Control Your Results Level 1 ID______________ Level 2ID_______________ Accepting Patient Results? Reason Controls’ range of expected results.UNIT: Total and Direct Bilirubin (continued) IV.Clinical Chemistry Lab Manual . Show at least one example calculation for indirect bilirubin on the back of this page. The bilirubin curve must accompany the results sheet. 3. Indirect Bilirubin Identification Concentration (units) Control 1 ____________ Control 2 ____________ Calculation formula(s) and examples NOTES: 1. 2. In control? Yes / No F 120 C MLAB 2401 .

Describe the relationship between hemoglobin and bilirubin. (3 points) 4. The conjugation of bilirubin occurs at what location? 5. answer the following questions.UNIT: Total and Direct Bilirubin (continued) Name Date Study Questions Instructions: Legibly write your answers in the space provided. Using lecture notes. Unless otherwise indicated. each question is worth one point. During the conjugation process. reading assignments and information presented in this lab.Clinical Chemistry Lab Manual C F 121 . How are bilirubin values used? 8. List three acceptable adjectives or names for the bilirubin molecule before it is conjugated. What different reagent(s) are needed for the total bilirubin procedure? What is (are) their purpose(s)? (2 points) MLAB 2401 . (½ point each. 3. (3 points) 7. List reagents needed to perform the direct bilirubin procedure. Free bilirubin appears in the plasma attached to . 1. bilirubin will be combined with what substance? 6. List three acceptable adjectives or names for the bilirubin molecule after it has been conjugated. 2. 2 points total) 9.

How is urobilinogen related to bilirubin? 11. Associate the different basic types of jaundice with increased levels of bilirubin by completing the following chart. What special procedures are needed in the handling of bilirubin samples? Why? 12.UNIT: Total and Direct Bilirubin (continued) 10. What components are in Diazo Reagent? 13. (3 points) increased bilirubin levels indirect direct total seen / often associated in this type of jaundice F 122 C MLAB 2401 .Clinical Chemistry Lab Manual .

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