1.0 INTENDED USE
This
reagent is intended for the quantitative determination of
direct
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 and Direct Bilirubin methods use
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 bilirubin reacts with diazo in the absence of DMSO. The intensity of the color produced is
directly proportional to the amount of direct bilirubin concentration present
in the sample.
3.3 CLINICAL SIGNIFICANCE
An elevation in body bilirubin because of increased
formation or retention of the pigment may cause a jaundiced condition
characterized by high levels of serum bilirubin and a yellowish pigmentation of
the skin. Jaundice may be classified as
prehepatic, hepatic or posthepatic depending upon the principal cause of the
condition. (10.10) Determination of both
total serum bilirubin and direct bilirubin (water-soluble bilirubin derivates
such as mono and diglucuronides) may help in the differential diagnosis of
jaundice.
3.0 SPECIMEN COLLECTION AND HANDLING
Serum is
the required sample for Biotron Diagnostics Direct Bilirubin Test.
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh, clear, unhemolyzed, 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 5-100 µl. Call King's
technical service department at 1-800-262-8655 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. (10.9) 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 within 4 immediately,
store the sample properly using the guidelines above.
4.0 MATERIALS (2 X 125 ml)
(4 X 125 ml)
Reagents necessary for the determination of direct
bilirubin are included in the kit.
4.1 REAGENT
4.1.1 Direct
bilirubin reagent contains:
sulfanilic acid 32
mM
hydrochloric acid 165
mM
4.1.2 Sodium
nitrite reagent contains:
Sodium nitrite ³ 29 mM
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 by combining 3 ml of
direct bilirubin reagent and 1 drop of sodium nitrite reagent. Mix well before using.
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 24 hours at room
temperature when stored in an amber bottle (or 10 days when refrigerated at
2-8° C.)
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance at 555 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 555 nm.
4.5.3 Test tubes
capable of holding 2 ml.
4.5.4 Pipettes
capable of delivering 1 ml, 100 µl.
4.5.5 Timer for 1
or 5 minute incubation.
4.5.6 Calibrator
/Control
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 555
nm
Temperature 18
‑ 26° C or 37° C
Pathlength 1.0
cm
Mode endpoint
Reaction time 5 minutes
at 18 ‑ 26° C, or
1
minute at 37° C
Sample volume 100
µl
Reagent volume 1.0
ml
Total volume 1.1
ml
Sample to reagent
ratio 1/10
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 555 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 direct 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.
5.4.2. Label test
tubes "Calibrator Test" and "Patient Test."
5.4.3. Label test
tubes "Calibrator Blank" and "Patient Blank."
5.4.4. Add 1.0 ml of
working reagent to the test tubes labeled "Calibrator Test" and
"Patient Test."
5.4.5. Add 1.0 ml of
direct bilirubin reagent to the test tubes labeled "Calibrator Blank"
and "Patient Blank."
5.4.6. At timed
intervals add 0.1 ml (100 µl) of patient sample to the "Patient Test"
and "Patient Blank" tubes, add 0.1 ml (100 µl) of calibrator sample
to the "Calibrator Test" and "Calibrator Blank" tubes. Mix well.
5.4.7. Incubate all
test tubes at 18 ‑ 26° C (room temperature) for 5 minutes or at 37° C for
1 minute.
5.4.8. Determine the
absorbance of all test tubes at 555 nm using distilled water as the reagent
blank.
5.5 PROCEDURE NOTE
5.5.1 Several drugs
which may cause elevated bilirubin levels are acetaminophen, chlordiazepoxide,
novobiocin and acetohexamide.
5.5.2 For pediatric
samples with bilirubin above 3.0 mg/dl, the sample should be diluted with an
equal volume of deionized water. Reassay
the sample and multiply the results by two.
5.6 CALCULATION AND RESULTS
A
‑ Ab
Bilirubin
(mg/dl) = ---‑‑‑--‑‑‑‑‑----- X
bilirubin value of calibrator
Ac ‑ Acb
A = absorbance of "patient test"
Ab = absorbance of "patient blank"
Ac = absorbance of "calibrator test"
Acb = absorbance of "calibrator
blank"
Example:
0.05
‑ 0.01
Bilirubin
concentration = ‑‑‑‑‑‑‑‑‑‑‑----‑
X 5.0 mg/dl = 0.83 mg/dl
0.25
‑ 0.01
A = 0.05,
Ab = 0.01, Ac = 0.25, Acb = 0.01
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: 0.0 ‑ 0.5 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
Due to the photodegratory nature of bilirubin, it is
important that all samples and standards be protected from direct light. 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 direct 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, 30 replicates of 2 control sera were run.
Mean
(mg/dl) SD (mg/dl) CV (%)
0.96 ± .007 0.69
1.26 ± .016 1.3
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 (%)
0.95 ± 0.007 0.77
1.20 ± 0.030 2.5
9.2 CORRELATION
A correlation study was done on the Gilford Stasar III
(registered trademark of Gilford Instruments) comparing this method and a
similar direct bilirubin method.
Number Regression Equation Correlation
of
Samples y=Biotron,
x=Comparative Coefficient
25 y = 1.04 x - 0.03 0.992
9.3 RECOVERY
In this study, known bilirubin protein standards of
varying concentration are added to a pool serum. With this method, recovery is in the range of
94‑103%.
9.4 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., Clin. Microchem. J., 15(1970)231.
10.6 Jendrassik,
L., and Grot, P., Vereinfachte Photometrische Methoden Zur Bestimmung des
Bluebilirubins, 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, 3rd ed., Washington DC, AACC Press
(1990).
10.9 Martinek,
R.G. Clin Chem Acta 13,161(1966).
10.10 Routh, J.I.,
Liver Function, Fundamentals of Clinical Chemistry, Edited by N.W. Tietz,
Philadelphia, Saunders (1976) 1026.
10.11 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
Rev
10/99