SUMMARY AND EXPLANATION

Increased serum bilirubin levels produce the clinical condition known as jaundice. Two forms of bilirubin can be responsible for jaundice - direct/conjugated or indirect/ unconjugated. The liver converts the unconjugated form so that it can be excreted. In obstructive jaundice and liver disease, this excretion is impaired and an elevated direct fraction occurs in the serum. The liver of newborns is not fully developed at birth, and therefore, is unable to convert the unconjugated form to the conjugated form for excretion. For this reason, increased indirect bilirubin is an important measurement for newborns (1).

Several methods for determining direct bilirubin have been described leading to much uncertainty about the method of choice. Direct bilirubin was originally defined by Van den Bergh and Miller (2) as that portion of the bilirubin in serum which reacts with diazo reagent in the absence of an accelerator. One of the most widely used methods for determining direct bilirubin has been that of Jendrassik-Grof (3), in which a 5 minute color development is employed. The EAGLE DIRECT BILIRUBIN PROCEDURE shows excellent agreement with this method while offering several improvements including faster reaction time, a more stable working reagent, better precision and direct calibration through the use of a stable aqueous calibrator.

PRINCIPLE

Conjugated (direct) bilirubin couples with a diazotized sulfanilic acid to form a colored complex that is measured 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 (4).

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2930 provides:

DIRECT BILIRUBIN REAGENT - (Cat. No. 2931)

REACTIVE INGREDIENTS:

0.032 M Sulfanilic Acid. Stabilizer added.

BILIRUBIN ACTIVATOR - (Cat. No. 2902)

REACTIVE INGREDIENTS:

60mM Sodium Nitrite. Stabilizer added.

PRECAUTIONS:

Exercise normal laboratory care in handling reagents. DIRECT BILIRUBIN REAGENT can cause irritation. In case of contact with eyes, skin or clothing, wash with large amount of water.

STORAGE AND STABILITY:

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

DETERIORATION:

All reagents should be clear and colorless solutions. Turbidity or formation of a precipitate would indicate deterioration. Additionally, development of a dark yellow solution in the ACTIVATOR REAGENT indicates that the reagent should not be used.

INSTRUMENTS

Uses 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.

SAMPLE STORAGE:

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

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

oxyhemoglobin  (5).

2. Lipemic sera will yield falsely elevated results. A specimen blank is recommended to correct for turbidity.

3. Young et al have reviewed drug effects on serum bilirubin assayed

by diazo methods (5).

PROCEDURE

MATERIALS PROVIDED:

DIRECT BILIRUBIN REAGENT (Cat. No. 2931) and BILIRUBIN ACTIVATOR (Cat. No. 2902).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.05 mL micropipetor

2. 1.0 mL pipettor or dispenser

3. TOTAL BILIRUBIN REAGENT SET (Cat. No. 2900)  provides sufficient CALIBRATOR to allow for calibration of the DIRECT BILIRUBIN PROCEDURE.

REACTION CONDITIONS:

Wavelength 540 nm

Incubation Time 3 minutes

Incubation Temperature Ambient

Sample Volume 0.05 mL

Direct Reagent Volume 1.0 mL

Activator Volume 0.02 mL

Total Volume 1.02 mg/dL

Low Normal 0.0 mg/dL

High Normal 0.5 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 DIRECT BILIRUBIN REAGENT needed. MIX WELL! Use working reagent within 24 hours of preparation if stored at room temperature. May be stored at 2 - 8ºC and used within 7 days of preparation. Discard if a dark yellow color develops.

MANUAL PROCEDURE:

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

2. Add 0.02 mL BILIRUBIN ACTIVATOR REAGENT to each tube. (Same ACTIVATOR REAGENT as used for Total.) MIX WELL! Omit this step when using Working Reagent.

3. At timed intervals, place 0.05 mL specimen into appropriately labeled tubes. Use D-H2O as specimen for Reagent Blank, Treat Calibrator exactly as specimen and use as supplied.

4. MIX WELL! Let stand for EXACTLY 3 MINUTES.

5. Adjust instrument to zero absorbance at 540 nm using Reagent Blank. Read and record absorbance values.

NOTE: For direct read-out instruments, set value of Calibrator as Concentration of Standard and read Unknown direct in concentration.

PROCEDURAL NOTES:

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).

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:

Timing of Unknowns must be exact to assure precise measurement of the direct fraction. The absorbance reading will continue to increase slowly due to the presence of the indirect fraction. The timing of the calibrator is not critical as the reaction is complete within 3 minutes.

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 EAGLE TOTAL BILIRUBIN REAGENT SET (Cat. No. 2900) 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:

                                                       Abs. Unk.

   Unk. Conc. (mg/dL)      =    -----------------     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 Direct Bilirubin concentratrion 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 and INFANTS (over 1 month):       <<0.5 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

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.

MEAN / ST. DEV. / %CV

WITHIN RUN

Normal 3.70 / 0.067 / 1.82

RUN TO RUN

Normal 3.62 / 0.100 / 2.76

SPECIFICITY:

A comparison of this DIRECT BILIRUBIN PROCEDURE with a commercial procedure using a Jendrassik-Grof modification showed a 99.7% correlation for 25 samples in the abnormal range.

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. 1040, W.B. Saunders Co., 1976.

2. Van den Bergh, A. A.H., and Miller, P., Biochem, Z 77, 90 (1916).

3. Jendrassik, L., and Grof, P., Biochem, Z 297, 81 (1938).

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

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

6. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin. Chem., 21, 226D (1975).