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

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

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)

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

Bilirubin Metabolism

Normal Values The sum of the direct and indirect forms is termed total bilirubin Adults: 0.2-1.0 mg/dl Conjugated: 0.0-0.2 mg/dl Unconjugated: 0.2-0.8 mg/dl Infants: Normal values depend on the age of the baby in hours and whether the baby was premature or full term

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

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

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

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

Types of Jaundice

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

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

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

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

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

Methods of Determination Spectrophotometric for neonatal bilirubin: Up to 21 days The absorbance of the sample is measured using a two-filter (455-575 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

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)

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).

**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#1 0.05 mL Ref and 0.15 mL water T#2 0.10 mL Ref and 0.10 mL water T#3 0.20 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).

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.

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.

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.

References Clinical Chemistry Lab Manual. Unit: Total and Direct Bilirubin. http://www.2ndchance.info/dxme-BilirubinUrine-defDirect.pdf