The following is a general procedure for use on a manual
spectrophotometer.
Procedure Conditions
Wavelength 540
nm
Temperature 37°
C
Mode Endpoint
Sample to Reagent Ratio 1:70
TEST PROCEDURE
(1)
A. Label
reagent tubes, as "BLANK", "STANDARD" and
"PATIENT".
B. Add 1 ml of
BUN reagent to each reagent tube.
C. Add 0.5 ml
of color developer solution to each reagent tube. Mix well.
D. Add 50 ml of BUN standard to the "STANDARD" tube. Mix well.
E. Add 50 ml of patient serum to the "PATIENT" tube. Mix well.
F. Incubate
reagent tubes at 37° C for exactly 5 minutes.
G. Add 2 ml of
diluent to each reagent tube. Mix well.
H. Wipe the
reagent tubes clean with a lint‑free tissue.
I. Place the
"BLANK" reagent tube in the test well and adjust the photometer to
zero absorbance.
Calculation Patient BUN concentration =
absorbance
of "PATIENT" sample
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑---------------------- X
value of standard
absorbance
of "STANDARD" sample
EXPECTED VALUES
NOTE: 1. (a) Bilirubin
has no effect if present in concentrations of less than 20 mg/dl and hemoglobin
has no effect if present in concentration of less than 150 mg/dl.
(b) Sulfanilamide and its derivative produce
false elevations.
INTENDED USE
Quantitative determination of Blood
Urea Nitrogen (BUN) in serum.
SUMMARY AND EXPLANATION
The two commonly used methods for measuring urea nitrogen
are the diacetyl reaction (1) and urease phenol and hypohalite reaction (2). In
1975 Jung and Biggs (3) developed a method to directly measure urea
nitrogen. This method does not require
elevated temperatures for color development, uses nonvolatile and stable
reagents and has specificity comparable to that of the diacetyl reaction. The
Biotron Diagnostics method is the modification of this new colorimetric
reaction for end point measurement of urea nitrogen.
TEST PRINCIPLE
Urea reacts with o‑phthalaldehyde to form
isoindoline which reacts with
Naphthylethylenediamine to form a chromophore whose color
intensity is directly proportional to the concentration of urea nitrogen.
MATERIALS PROVIDED
BUN reagent 132
ml
Color developer 66
ml
Diluent 264
ml
Standard/Control/Calibrator
For In Vitro Diagnostic use.
BUN Reagent ‑ contains 0.6% o‑phthalaldehyde,
9.5% of concentrated sulfuric acid and a surfactant.
Color developer ‑ contains 0.5%
naphthylethylenediamine, 6% of boric acid and a surfactant.
Diluent ‑ contains 0.1% of concentrated sulfuric
acid.
CAUTION! Do not take these reagents internally or allow
them to come in contact with the body.
STORAGE
Store reagents at room temperature (18‑26° C). All reagents are stable till the expiration
date stated on the label when stored at room temperature.
ADDITIONAL MATERIALS REQUIRED
Spectrophotometer, colorimeter or blood analyzer
Pipetting device
Reagent Tubes or Cuvettes
37° C heat source
SAMPLE PREPARATION
Freshly drawn, fasting serum is the specimen for Biotron
Diagnostics BUN test.
EXPECTED VALUES
(4)
Normal BUN range is 8‑23 mg/dl.
The above range is intended as a guide. Each laboratory should establish its own
normal range.
PERFORMANCE
(5)
1. Precision ‑
The precision study was done by
(a) repetitive assay
(N=20) of normal serum specimen. This
assay yielded a mean of 12.4 mg/dl, a standard deviation of 0.51 mg/dl and a
coefficient of variation of 4.1%.
(b) repetitive assay
(N=20) of abnormal serum specimen. This
assay yielded a mean of 55.2 mg/dl, a standard deviation of 1.5 mg/dl and a
coefficient of variation of 2.7%.
(c) 8 day reproducibility
study. A pool serum specimen with
a mean of 15.7 mg/dl yielded a standard deviation of 0.58 mg/dl and a
coefficient of variation of 3.7%.
2. Accuracy ‑
The accuracy study was done by
(a) running 23 serum specimens on SMA 12/60
(registered trademark of Technicon Corporation) procedure and Biotron
Diagnostics method on Gilford Stasar III (registered trademark of Gilford
Instrument). The study yielded a
regression equation of Biotron = 1.036 x
reference method - 0.85 and a correlation of 0.98.
(b) adding known BUN aqueous standards of
varying concentration with Biotron
Diagnostics method. Recovery was
in the range of 97.5% to 101%.
LIMITATIONS OF THE PROCEDURE
This procedure is linear through 70 mg/dl beyond which
the specimen should be diluted 1:2 with deionized water. Reassay the specimen and multiply the result
by 2.
QUALITY CONTROL
Standard practice for quality control should be applied to this system. Commercially available lyophilized controls can be used. Daily quality control must fall within 2 standard deviations of the established value. If correlation is not obtained and repetition of the assay excludes errors in technique, the following steps should be taken:
1. Calibrate
the instrument according to manufacturer's instructions.
2. Check the
expiration date of the reagent package.
3. Check the
cleanliness of the reagent tube.
4. Contact
Biotron Diagnostics Technical Services Department.
REFERENCE
1. Henry,
R.J., Clinical Chemistry, Principles and Techniques, Harper and Row, N.Y.,N.Y.
1967, p.266.
2. Marsh,
W.H., Fingerhut,B. and Millard, H. "Clinical Chemistry" 11:624, 1965.
3. Jung, D. et
al "Clin Chem" 21:1136, 1975.
4. Tietz, N.W.
"Fundamentals of Clinical Chemistry" W.B. Saunders, Philadelphia,
1970.
5. Kin
Diagnostics Laboratory Data, Indianapolis, IN, 1981.