1.0 INTENDED USE
This reagent is intended for
the quantitative determination of urea nitrogen (BUN) in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Urea has been determined by
the direct method (10.2) where urea condences with diacetyl to form a chromagen
and an indirect method where ammonia is measured as a product of Urease action
on urea. (10.3)
The liberated ammonia has
been measured using Nessler’s reagent (10.4) and by the Berthelot reactiion. (10.5) Talke and Schubert introduced a
totally enzymatic procedure in 1965 initializing Urease and Glutamate
Dehydrogenase. (10.6) The
present procedure is based on a modification of their method.
2.2 TEST PRINCIPLE
In the Talke and Schubert
procedure urea is first hydrolyzed by urease to give ammonia and carbon dioxide
as in the equation:
Urease
Urea + H2 ----------> 2 NH3
+ CO2
In the second step of the
process the ammonia produced in the first reaction reacts with 2-oxoglutarate
and NADH in the presence of glutamate dehydrogenase(GLDH)
to yield glutamate and NAD.
GLDH
NH3 + 2-oxoglutarate + NADH +
H+ --------> L-glutamate +NAD+ +H2O
Urea is hydrolyzed by Urease
to produce ammonia and water. The
liberated ammonia reacts with a-Ketoglutarate
in the presence of NADH to yield glutamate.
An equimolar quantity of NADH undergoes oxidation during the reaction
resulting in a decrease in absorbance that is directly proportional to the urea
nitrogren concentration in the sample.
2.3 CLINICAL SIGNIFICANCE
(10.1)
Determination of urea
nitrogen in serum is widely used as a screening test for renal function. When used in conjunction with the
determination of creatinine in serum it is helpful in the differential
diagnosis of the three types of azotemia; prerenal, renal and post-renal.
3.0 SPECIMEN COLLECTION AND
HANDLING
3.1 PATIENT PREPARATION
No special patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh, clear, unhemolyzed
serum is the preferred specimen. The
serum should be promptly separated from the clot. Fluoride is a known inhibitor of urease. Therefore anticoagulants containing fluoride
should be avoided. (10.8)
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 5-25 µl. Call Biotron'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
Samples may be stored
refrigerated (2-8°C) for 3-5 days or frozen (-20°C) for several months.
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 BUN are
included in the kit.
4.1 REAGENT
BUN working reagent contains:
alpha-ketoglutarate 4.0 mM
ADP 2.0
mM
NADH 0.28
mM
urease >15,000
U/L
glutamate dehydrogenase (bovine) >1667 U/L
sdium azide 0.2%
buffer pH 7.6+/- 0.1
4.1.1 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.
Sodium azide may
react with lead and copper plumbing to form highly explosive metal azides. Upon
disposal, flush with large amounts of water.
4.3 REAGENT PREPARATION
The working reagent is ready
to use as is.
4.4 REAGENT STORAGE AND
STABILITY
Store
reagents in refrigerator. Protect from light and
freezing. Unopened reagents are stable at 2-8° C (refrigerated) until the
expiration date stated on the labels.
The working reagent is stable for 14 days at 2°-8° C and 3 days at
18-26° C.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 Spectrophotometer or colorimeter capable of reading absorbance at
340 nm.
4.5.2 1 cm cuvettes or a flow cell capable of
transmitting light at 340 nm.
4.5.3 Test tubes and
pipettes.
4.5.4 Timer .
4.5.5 Constant temperature source which can be adjusted to 37° C.
4.5.6 Normal and abnormal controls for quality control.
5.0 TEST PROCEDURE
The following is a general procedure for use on a manual
instrument.
Application procedures for
use on automated analyzers are available. Contact Biotron’s Technical Service
Department for specific information.
5.1 PROCEDURE CONDITIONS
Wavelength 340
nm
Temperature 37°
C
Pathlength 1.0
cm
Mode endpoint
Lag time 30
sec
Read time 60
sec
Sample to reagent ratio 1:100
Reaction direction decreasing
5.2 INSTRUMENT
Any instrument capable of
reading absorbance accurately with a sensitivity of 0.001
absorbance at 340 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
Use Biotron's serum based
calibrator. The BUN assay is calibrated
by referencing the absorbance of the unknown sample to the absorbance of the
calibrator.
5.4 PROCEDURE
The following procedure is a
general procedure for use on a manual instrument.
5.4.1 Prepare the required volume of working reagent (see 4.3 Reagent
Preparation Section.)
5.4.2 Adjust the absorbance reading at 340 nm on the spectrophotometer to
0.000 using distilled water as the blank.
5.4.3 Pipette 1.0 ml of working reagent into test tubes. Determine the
absorbance of the working reagent at 340 nm. This should be at least 1.0.
5.4.4 Into separate test tubes pipette 10 µl of calibrator or serum to be
assayed.
5.4.5 Incubate at 37° C for 30 seconds. Record the absorbance reading A1.
5.4.6 Incubate for another 1 minute at 37° C. Record the absorbance (A2).
5.4.7 Record the change in absorbance DA (A2-A1).
5.5 CALCULATION AND RESULTS
DA
Urea = ------------ X concentration of calibrator
DAs
DA = change in absorbance of sample
DAs = change in absorbance of the calibrator
Example:
0.165
Urea = ---------- X 30 mg/dl = 13.3 mg/dl
0.372
with DA = 0.165, DAs =0.372, concentration of
calibrator = 30 mg/dl
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES
The range of expected values is: 8-26 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
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
Urease is specific for urea, however ammonia
contamination will seriously affect the results obtained using the system.
Analysis should not be performed in close proximity to a urinalysis laboratory
or in a laboratory using cleaning supplies containing ammonia.
A summary of the influence of
drugs of clinical laboratory tests may be found by consulting Young D.S., Et. Al.(10.7). Severely icteric,
hemolytic, or lipemic samples require the use of a sample blank which may be
prepared using 10 µl of sample and 3 ml of deionized water.
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 BUN 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 QC data.
9.0 PERFORMANCE
CHARACTERISTICS
9.1 PRECISION
The estimates of precision
shown below were obtained from assays of human control serum.
Within-Run
Mean (mg/dl) SD
(mg/dl) CV (%)
10.1 ±
0.3 3.1
68.4 ±
0.6 1.4
144.3 ±
1.9 1.3
Between-Run
Mean (mg/dl) SD
(mg/dl) CV (%)
10.3 ±
0.5 4.6
69.3 ±
0.9 1.3
145.1 ±
2.3 1.6
9.2 CORRELATION
A correlation study was done
by running 107 specimens, ranging from 5 to 139 mg/dl, with a similar
comparative method.
Number Regression
Equation Correlation
of Samples y=Biotron,
x=Comparative Coefficient
107 y
= 1.05x - 0.2 0.999
9.3 LINEARITY
This procedure is linear
through 150 mg/dl beyond which the specimen should be diluted 1 to 1 with
deionized water. Reassay the specimen
and multiply the results by 2.
10.0 REFERENCES
10.1 Tietz, N.W., Fundamentals of Clinical Chemistry,
10.2 Fearon, W.R., Biochem J. 331:902(1939).
10.3
10.4 Gentzkow, C.J., J. Biol. Chem. 143:531(1952).
10.5 Fawcett, J.K., Scott, J.E., J. Clin. Path.
13:156(1960).
10.6 Talke, H., Schubert, G.E., Klin. Wschr.
43:174(1965).
10.7 Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia W.B.
Saunders, p.991(1976).
10.8 NCCLS document “Protection of Laboratory Workers from Infectious
Disease Transmitted by Blood, Body Fluids, and Tissue”, 2nd Ed.(1991).
10.9 Young D.S., et al, Clin. Chem. 21:1D(1975).
10.10 NCCLS document “Evaluation of Precision Performance of Clinical Chemistry Devices:, 2nd Ed.(1992).