1.0 INTENDED USE
This reagent is intended for the quantitative determination
of
total bilirubin in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
In 1883 Ehrlich introduced
the diazo reaction for the detection of bilirubin (10.1). In 1913 Van den Bergh
and Snapper (10.2) applied the diazo reaction to serum after
deproteinization. In 1916 Van den Bergh
and Muller (10.3) discovered the direct and indirect reading of bilirubin in
serum. In 1937 Malloy and Evelyn (10.4) adapted the bilirubin procedure to the
photoelectric colorimeter. The Biotron
Diagnostics Total Bilirubin method uses DMSO based on modification of
Walters and Gerard (10.5). The method is sensitive, accurate and easy to
perform. It compares very favorably with
Malloy and Evelyn (10.4) and Jendrassik and Grot (10.6).
2.2 TEST PRINCIPLE
Sulfanilic acid reacts with
sodium nitrite to produce diazotized sulfanilic acid (diazo). Direct and indirect bilirubin couple with
diazo to produce azobilirubin in the presence of dimethyl sulfoxide
(DMSO). The intensity of the color
produced is directly proportional to the amount of total bilirubin
concentration present in the sample (at 546 nm.)
2.3 CLINICAL SIGNIFICANCE
(10.9)
Total serum bilirubin is
elevated in cases of obstructive jaundice, hepatitides, and cirrhosis. Elevations will occur when there is excessive
destruction of hemoglobin.
3.0 SPECIMEN COLLECTION AND
HANDLING
3.1 PATIENT PREPARATION
No special patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh, clear, fasting serum is the preferred specimen.
Fasting avoids lipemic
interference. Hemolyzed samples may
produce falsely low values.
Use a standard venipuncture tube to draw patient sample.
The amount of sample required
will depend on the analyzer used. The
amount of serum required is in the range of 5-200 µl. Call Biotron's technical service department
at 1-800-595 8766 for the recommended sample volume for your analyzer.
Record the patient's name,
date and time of sample collection and preparation.
3.3 SPECIMEN STORAGE
Specimens must be stored away
from direct light as bilirubin is subject to photodegradation. Serum samples may be stored for 2 hours at
room temperature, 12 hours when refrigerated and 3 months when frozen.
(10.10) Frozen samples should be thawed
at room temperature and mixed completely before analysis. Thawed samples should not be refrozen.
It is recommended that
testing be done as soon as possible after sample collection and
preparation. If testing cannot occur
immediately, store the sample properly using the guidelines above.
4.0 MATERIALS (2 X 125
ml)
Reagents necessary for the
determination of total bilirubin are included in the kit.
4.1 REAGENT
4.1.1 Total bilirubin reagent contains:
Sulfanilic acid 32
mM
hydrochloric acid 165
mM
dimethyl sulfoxide 7000
mM
4.1.2 Sodium nitrite reagent contains:
Sodium nitrite ³ 29 mM
4.1.3 Standard/Control/Calibrator
4.2 WARNINGS AND PRECAUTIONS
For In
Vitro Diagnostic Use. Not for Internal use in Humans
or Animals. In Vitro Diagnostics
reagents may be hazardous. Avoid
ingestion and skin or eye contact.
4.3 REAGENT PREPARATION
The working reagent is
prepared adding 1 drop of sodium nitrite for each 3 ml of total bilirubin
reagent. Mix well before using.
Record the data and time of reconstitution.
4.4 REAGENT STORAGE AND
STABILITY
When stored at 18°-26°C
unopened reagents are stable until the expiration date printed on the
label. The working reagent is stable for
8 hours at 18°-26°C or 30 days at 2-8°C when stores tightly capped in an amber
bottle.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 Spectrophotometer or colorimeter capable of reading absorbance at
550-560 nm.
4.5.2 1 cm cuvettes or a flow cell.
4.5.3 Test tubes capable of holding 3 ml.
4.5.4 Pipettes capable of delivering 3 ml and 200 µl.
4.5.5 Timer for a 1 or 5 minute incubation.
4.5.7 Normal and abnormal control for quality control.
5.0 TEST PROCEDURE
The following is a general procedure for use on a manual
instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 550-560
nm
Temperature 18
- 26°C or 37°C
Pathlength 1.0
cm
Mode endpoint
Reaction time 5
minutes at 18 - 26° C
1
minute at 37° C
Sample volume 200
µl
Reagent volume 3
ml
Total volume 3.2
ml
Sample to reagent ratio 1/15
5.2 INSTRUMENT
Any instrument capable of
reading absorbance accurately with a sensitivity of 0.001
absorbance at 550-560 nm may be used.
The band width should be 10 nm or less, stray light 0.5% or less, and
the wavelength accuracy within 2 nm.
5.3 CALIBRATION
The total bilirubin assay is calibrated by
referencing the absorbance of the unknown sample to the absorbance of the
calibrator.
5.4 PROCEDURE
5.4.1 Prepare the required volume of working reagent. (See 4.3 Reagent
Preparation section.)
5.4.2 Into appropriately labeled tubes, add 3 ml of working reagent.
5.4.3 Add 0.2 ml (200 µl) of sample to the appropriate tube.
5.4.4 Incubate all test tubes at 18 - 26°C (room temperature) for 5
minutes or at 37°C for 1 minute.
5.4.5 Determine the absorbances of the all test tubes at 550-560 nm using
the working reagent as the reagent blank.
5.5 PROCEDURE NOTE
The color of the final mixture is stable for 30 minutes.
5.6 CALCULATION AND RESULTS
A
Bilirubin (mg/dl) = ----
X bilirubin value of calibrator
Ac
A = absorbance of
unknown, Ac = absorbance of calibrator
Example:
0.230
Bilirubin concentration = ---------
X 5.0 mg/dl = 4.2 mg/dl
0.275
with A = 0.230, Ac = 0.275 and bilirubin value of
calibrator = 5.0 mg/dl.
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.7)
The range of expected values is: 0.2 - 1.2 mg/dl
These values are suggested
guidelines. It is recommended that each
laboratory establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES
(10.11)
Each laboratory should
establish low and high values beyond which the patient would require immediate
attention by a physician. If a
"medical alert value" is reached, always repeat the test to confirm
the result and notify a physician if the result is confirmed.
6.3 LIMITATIONS OF PROCEDURE
A comprehensive list of drugs
and other substances that affect total bilirubin is given by Young. (10.8)
7.0 QUALITY CONTROL
Standard practice for quality
control should be applied to this system.
Commercially available lyophilized controls can be used to monitor the
daily acceptable variations. Normal and
abnormal controls should be assayed at the beginning of each run of patient
samples, whenever a new reagent or a different lot number is being used, and
following any system maintenance.
A satisfactory level of
performance is achieved when the analyte values obtained are within the
"acceptable range" established by the laboratory.
8.0 CALIBRATION PROCEDURES
The total bilirubin assay is
calibrated by referencing the absorbance of the unknown sample to the
absorbance of the calibrator. Refer to
your instrument manual for more details.
Calibration is required with
the use of a new lot of reagent, any system maintenance or whenever indicated
by quality control data.
9.0 PERFORMANCE
CHARACTERISTICS
9.1 PRECISION
The estimates of precision
shown below were obtained from assays of human control serum.
Within-Run: In this study, 15 replicates of 2 control sera
were run.
Mean (mg/dl) SD
(mg/dl) CV (%)
1.42 ±
0.04 2.82
5.84 ±
0.06 1.05
Between-Run: In this study, 5
runs were made, each run consisting of 5 replicates of 2 control sera.
Mean (mg/dl) SD
(mg/dl) CV (%)
1.44 ±
0.05 3.40
5.88 ±
0.08 1.33
9.2 CORRELATION
A correlation study was done
on the Technicon RA-500 system at 37° C comparing this method and a similar
total bilirubin method. The samples
range between 0 mg/dl and 23.8 mg/dl.
Number Regression Equation Correlation
of Samples y=Biotron,
x=Comparative Coefficient
40 y
= .978 x + .031 0.999
9.3 LINEARITY
This procedure is linear
through 20 mg/dl beyond which the specimen should be diluted with an equal
volume of deionized water. Reassay the
specimen and multiply the results by 2.
10.0 REFERENCES
10.1 Ehrlich, P., Klin Med, 45:721, 1883.
10.2 Van den Bergh, AAH, and Snapper, J., Dtsch Arch Klin Med, 110:540,
1913.
10.3 Van den Bergh, AAH, and Muller, P., Biochem Z, 77:90, 1916.
10.4 Malloy, H.T., and Evelyn, K.A., J Biol Chem, 119:481, 1937.
10.5 Walters, M., Gerard, H., Clinical Microchem Journal, 15(1970)231.
10.6 Jendrassik, L., and Grot, P., Biochem Z,297:81, 1938.
10.7 Henry, R. J., Clinical Chemistry: Principles and Techniques,
Harper and Row, N.Y., pp. 1059-60, 1974.
10.8 Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 13rd
ed., Washington DC, AACC Press (1990).
10.9 Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders
Co., Philadelphia, Pa, 1970, p.212
10.10 Martinek, R.G. Clin Chem Acta 13,161(1966).
10.11 G.J. Kost, "Critical Limits for Urgent Clinician Notification
at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5,
Rev
10//99