2.15: Bilirubin (Waters and Gerande – DMSO Method)

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RELATED READING: Chapters 27, 36. See Methods in CD-ROM for Bilirubin.

OBJECTIVES

Upon completion of this exercise, appropriate discussion, and related reading, the student will be able to:

Perform direct and total bilirubin analysis on 2 samples with 90% accuracy.
Explain the effect of free hemoglobin on bilirubin determinations in this method.

PRINCIPLE

Bilirubin will react with diazotized sulfanilic acid to yield a purple compound, azobilirubin. This product can be quantitated spectrophotometrically at 560 nm. This procedure is used to measure the soluble conjugated (direct-reacting) bilirubin alone or in combination with the less soluble unconjugated (indirect-reacting) bilirubin. To measure total bilirubin, a suitable solvent that allows for the measurement of both direct and indirect—reacting bilirubin is used. The unconjugated bilirubin concentration is the difference between direct-reacting and total bilirubin values. Care should be taken to protect samples from strong or prolonged light because bilirubin pigments are light sensitive.

MATERIALS

Total bilirubin reagent
Spectrophotometer
Direct-reacting bilirubin
Cuvettes
Sodium nitrite
Pipets
Standard
13 x 100 mm
Test Tubes
Controls
Samples

PROCEDURE

Total Bilirubin

Label a sufficient number of 13 x 100 mm test tubes as “Test” and “Blank” for each standard, control, and serum sample to be tested.
Add 3 mL of total bilirubin reagent to all “Blank” and “Test” tubes.
Using a Pasteur pipet, add 1 drop of sodium nitrite reagent to each “Test” tube.
At precise one minute intervals, add 200 \(\mu\)L of the appropriate standard/control/sample to each “Test” and “Blank” tube.
Mix well.
Incubate at room temperature for exactly 5 minutes.
Read the absorbance of the first sample against it’s own blank at 560 nm; repeat for each subsequent sample at 1 minute intervals.
Record your results on the data sheet.

Direct Bilirubin

Label a sufficient number of 13 x 100 mm test tubes as “Blank” and “Test” for the patient samples only.
Add 3 mL of direct bilirubin reagent to each “Blank” and “Test” tube.
Using a Pasteur pipet, add 1 drop of sodium nitrite reagent to each “Test” tube.
At precise one minute intervals, add 200 \(\mu\)L of serum to the “Blank” and “Test” tube for each sample and mix.
Incubate at room temperature for exactly 5 minutes.
Read the absorbance of the first sample against it’s own blank at 560 nm; repeat for each subsequent sample at 1 minute intervals.
Record your results on the data sheet.

OPTIONAL EXERCISE

Prepare red blood cells washed free of plasma. Resuspend the cells with distilled water to 5 times the original blood volume.
Add 0.05 mL of this hemolysate to one of the controls or samples run in the above procedure described for total bilirubin.
Record the results on the data sheet.

DATA SHEET, EXERCISE #15

NAME: ___________

DATE: ___________

RESULTS

	Absorbance (total)	Total Bilirubin (mg/L)	Absorbance Direct	Direct (Conjugated) Bilirubin (mg/L)	Indirect (Unconjugated Bilirubin (mg/L)
Standard
Normal Control
Abnormal Control
Sample #
Sample #

CALCULATIONS

Calculate the total bilirubin for the controls and serum samples. Calculate the direct and indirect bilirubin for the patient serum samples. Use the absorbance/concentration proportion method.

\[\text{Total bilirubin} = \text{conjugated (“direct”) bilirubin} - \text{unconjugated (“indirect”) bilirubin}\]

\[\text{Unconjugated bilirubin} = \text{Total} - \text{Direct bilirubin}\]

Optional Exercise

	Absorbance	Total Bilirubin (mg/L)
Hemoglobin-spiked sample
Unspiked sample (# )

Discussion Questions

What substances are known to cause interference with this method?
Why is it necessary to run a serum blank with each sample?
What is the purpose of the sodium nitrite in the reaction?
Is the type of blank employed in this exercise a reagent or sample blank?
What compound was employed in the “total” reagent to solubilize the unconjugated bilirubin? What other solubilizers were also used?