TOTAL BILIRUBIN PROCEDURE

Intended for the Quantitative Determination of Bilirubin in Serum

SUMMARY AND EXPLANATION

Bilirubin is a bile pigment formed by the degradation of hemoglobin released from old or damaged erythrocytes. Normally, about six grams of hemoglobin is released daily. This hemoglobin enters the cells of the reticuloendothelial system (spleen, liver and bone marrow) where it is converted to bilirubin. Bilirubin is then linked to albumin and transported to the liver where it is removed as a waste product. An increase in the formation or retention of bilirubin in the body results in jaundice and an increase in the serum level of bilirubin. This hyperbilirubinemia is classified as either prehepatic, hepatic or post-hepatic depending on the principal cause of the condition (1). Therefore, determination of the "Total" Bilirubin and its conjugated "Direct" fraction is important for the differential diagnosis of hyperbilirubinemia.

Two forms of bilirubin (conjugated / direct and unconjugated / indirect) have been identified in serum based on their behavior in the diazo reaction. This procedure measures a "Total" bilirubin involving the coupling of both bilirubin fractions with diazotized sulfanilic acid to form a colored complex (azobilirubin) that can be read spectrophotometrically. The coupling reaction takes place in the presence of an accelerating agent to facilitate reaction of albumin-bound bilirubin with the diazo reagent (2).

PRINCIPLE

Bilirubin, both direct and indirect, couples with a diazotized sulfanilic acid in the presence of an accelerating agent to form a colored complex which is read at 540 nm. Calibration of the method is completed with the use of a pure aromatic amine which couples with the diazotized sulfanilic acid to yield a colored complex similar to that developed with serum bilirubin (3).

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2900 provides:

TOTAL BILIRUBIN REAGENT - (Cat. No. 2901)

REACTIVE INGREDIENTS:

0.032 M Sulfanilic Acid, Accelerator and Stabilizer added.

BILIRUBIN ACTIVATOR - (Cat. No. 2902)

REACTIVE INGREDIENTS:

0 mM Sodium Nitrite. Stabilizer added.

BILIRUBIN CALIBRATOR - (Cat. No. 2903)

REACTIVE INGREDIENTS:

0.346 mM N-1-naphthylethylenediamine. Stabilizer added. Equivalent to 5.0 mg/dL Bilirubin in described method.

PRECAUTIONS:

Exercise normal laboratory care in handling reagents. TOTAL BILIRUBIN REAGENT will cause irritation. In case of contact with eyes, skin or clothing, wash with large amount of water. DO NOT PIPET BY MOUTH.

STORAGE AND STABILITY:

Store all reagents at 2 - 8ºC. Stable until stated expiration date on bottles.

DETERIORATION:

CALIBRATOR should be clear and colorless solution. Turbidity or formation of a precipitate would indicate deterioration. The TOTAL REAGENT will be clear to a pale yellow solution. Development of a pink color indicates deterioration and the reagent should not be used. Additionally, development of a dark yellow color in the ACTIVATOR REAGENT indicates that the reagent should not be used.

INSTRUMENTS

Use a suitable spectrophotometer or colorimeter calibrated at 540 nm.

SPECIMEN COLLECTION AND HANDLING

1. Fresh unhemolyzed serum or plasma may be used.

2. Separate the serum from the blood clot as soon as possible.

3. SPECIAL PRECAUTION: Before analysis, the serum should be stored in the dark. Avoid direct sunlight or white light exposure as this may decrease bilirubin values up to 50% in one hour.

4. Turbid or lipemic samples require a Serum Blank.

SAMPLE STORAGE:

Serum bilirubin is stable up to 7 days stored at 2 - 8ºC or may be frozen for up to  three months if protected from light exposure (1).

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Gross hemolysis will cause falsely low values due to the inhibition of oxyhemoglobin

PROCEDURE

MATERIALS PROVIDED:

TOTAL BILIRUBIN REAGENT (Cat. No. 2901), BILIRUBIN ACTIVATOR (Cat. No. 2902), BILIRUBIN CALIBRATOR (Cat. No. 2903).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.05 mL micropipetor

2. 1.0 mL pipettor or dispenser

3. Test tubes and rack

4. Timer

REACTION CONDITIONS:

Wavelength 540 nm

Filter Selection 525 - 550 nm

Incubation Time 90 seconds

Incubation Temperature Ambient

Sample Volume 0.05 mL

Total Reagent Volume 1.0 mL

Activator Reagent Volume 0.02 mL

Total Volume 1.02 mL

Low Normal 0.1 mg/dL

High Normal 1.2 mg/dL

Calibrator Value 5.0 mg/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

PREPARATION OF WORKING REAGENT:

As an alternative to reagent preparation described in MANUAL PROCEDURE, a working reagent may be prepared. Add 1 drop of ACTIVATOR REAGENT to each 3.0 mL of TOTAL BILIRUBIN REAGENT needed. MIX WELL! Use working reagent within 24 hours of preparation if stored at room temperature. May be stored at 2 - 8º C and used within 7 days of preparation. Discard if a dark yellow color develops.

MANUAL PROCEDURE:

1. Dispense 1.0 mL of TOTAL BILIRUBIN REAGENT into tubes labeled Reagent Blank, Calibrator, Controls, Unknowns, etc.

2. Add 0.02 mL BILIRUBIN ACTIVATOR REAGENT to each tube. MIX WELL! (Omit this step if working reagent has been prepared).

3. Place 0.05 mL specimen into appropriately labeled tubes. Use D-H2 O as specimen for Reagent Blank. Treat Calibrator exactly as specimen and use as supplied.

4. MIX WELL! Let stand for at least 90 seconds.

5. Adjust instrument to zero absorbance at 540 nm using Reagent Blank.

6. Read and record absorbance values.

NOTE: For direct read-out instrument, set value of Calibrator as concentration of Standard and read Unknowns direct in concentration.

PROCEDURAL NOTE

When specimen size is limited, or specimen exceeds linearity of the procedure, reduce the volume and perform test as directed. Multiply result by appropriate factor (2 x for 0.025 mL or 5 x for 0.01 mL).

SERUM BLANK:

A Serum Blank may be employed for greater accuracy in the assay of turbid samples. Label an additional test tube as described in Step #1 in MANUAL PROCEDURE. Omit the BILIRUBIN ACTIVATOR REAGENT. Add equivalent amount of sample as used in total reaction. Read the absorbance of the Serum Blank tube with the instrument zero set as described in Step #5. The absorbance of the Serum Blank is subtracted from that of the Unknown specimen absorbance. This Corrected Absorbance is used in the calculation of results as the absorbance of the Unknown. In normal subjects the concentration of Total Bilirubin in serum can approach zero. If a blank correction is made for a turbid normal sample it is possible that the corrected value for Total Bilirubin may be near zero due to the turbidity of the sample.

STABILITY OF FINAL REACTION:

The test samples should be read within 30 minutes after color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in a range of 0 - 20.0 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use BILIRUBIN CALIBRATOR which is provided in the reagent set or other suitable calibration material for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control material (normal and abnormal) analyzed in the same manner as the Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

CALCULATIONS OF RESULTS

The following equation is used to determine Unknown concentration:

Unk. Conc. (mg/dL)   =

Abs. Unk.

---------------   X Cal. Conc. (mg/dL)

Abs. Cal.

EXAMPLE:

A 5.0 mg/dL Calibrator had an Abs. = 0.250 while the Unknown Abs. = 0.120. The Total Bilirubin concentration of the Unknown is:

0.120

--------     X 5.0 mg/dL    =   2.4 mg/dL

0.250

LIMITATIONS

1. Grossly hemolyzed samples can not be used.

2. Absorbance values of 1.0 will be noted for samples approaching 20.0 mg/dL. If this exceeds the capability of the instrument employed, reduce sample volume as described in PROCEDURAL NOTE.

EXPECTED VALUES (1)

ADULTS Up to 1.2 mg/dL

NEWBORN Up to 12.0 mg/dL

This range was established using samples from a clinically normal population. lt is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 20.0 mg/dL.

PRECISION:

Normal and abnormal sera were assayed 20 times each to establish within run precision and for 10 working days to establish run to run precision.

WITHIN RUN

MEAN / ST. DEV. / %CV

Abnormal 10.20 / 0.235 / 2.3

RUN TO RUN

Normal 0.51 / 0.018 / 3.5

Abnormal 10.24 / 0.25 / 2.4

SPECIFICITY:

A comparison of this TOTAL BILIRUBIN PROCEDURE with another widely used procedure showed a 99.95% correlation with a regression equation of y = 1.006X - 0.100.

SENSITIVITY:

This procedure has a sensitivity of 0.018 mg/dL per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, p. 1028, W.B. Saunders Co., 1976.

2. Walter, M. and Gerade, H., Microchem. Journ., 15, 231 (1970).

3. Bilissis, P.K., and Spear, R.J., Clin. Chem., 9, 552 (1963).

4. Shull, B., Clin. Chem., 26, 22 (1980).