CLS Laboratory
San Francisco State University
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory BUN Procedure #900.099
Section: Chemistry
Date of Implementation: June 6, 2015
Date of Revision: Stanbio BUN 0580 retired, April 4, 2015
BLOOD UREA NITROGEN STANDARD OPERATING PROCEDURE
Quantitative Colorimetric Determination of Urea Nitrogen in Serum, Plasma or Urine
Principle
Clinical
Significance
CPT Code
Specimen
Requirements
Urea is the primary waste product of protein catabolism. Urea is synthesized
in the liver from ammonia during the deamination of amino acids. Tissue
damage, starvation or a high protein diet accelerate the rate of urea
production. However, a healthy kidney is quite capable of excreting excess
urea. In patients with normal renal function, only extremely high protein
diets cause rises in plasma urea to levels above the normal reference range.
In patients with significantly increased tissue catabolism due to severe tissue
damage or starvation, plasma urea levels may rise above normal reference
ranges. Elevated plasma urea may be seen when renal function is impaired.
Therefore, the main use of the Blood Urea Nitrogen procedure is to evaluate
renal function.
The Stanbio BUN (0580) Procedure is based on diacetylmonoxime (DAM)
methodology. Acid-catalyzed condensation of urea with diacetylmonoxime
in the presence of thiosemicarbazide gives a red-purple chromogen which is
measured spectrophotometrically at 520 nm.
Urea is generated in the liver as a product of protein catabolism. The most
common cause of an elevated BUN is poor renal function. Greatly elevated
BUN may be indicative of renal failure. Elevated BUN with normal
creatinine concentrations may indicate a relative decrease of blood flow to
the kidney, as seen in heart failure or dehydration, without true renal injury.
A low BUN possesses little clinical significance but could be caused by liver
disorder, malnutrition, excessive alcohol consumption, and over hydration.
81793
Serum: The specimen should be analyzed as soon as possible following
collection to prevent hemolysis. Specimen minimum volume is 0.25 mL.
Plasma: Plasma may be used if anticoagulant is free of ammonium salts.
Specimen minimum volume is 0.25 mL.
Urine: Specimens should be preserved with Fluoride-Thymol. A dilution
must be performed. Add 19.0 mL of water to 1.0 mL of well mixed urine
specimen and mix well. Use 20.0 uL of the diluted urine for analysis and
multiply the observed value by a dilution factor of 20 to obtain the BUN
concentration of the urine specimen in mg/dL.
Sample Stability: Urea nitrogen in plasma or serum has been reported to be
stable for 24 hours at room temperature, several days under refrigeration
between 2-8 degrees Celsius, and 6 months when frozen at -20 degrees
Celsius.
Page 1 of 4
CLS Laboratory
San Francisco State University
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory BUN Procedure #900.099
Section: Chemistry
Date of Implementation: June 6, 2015
Date of Revision: Stanbio BUN 0580 retired, April 4, 2015
Specimen requirements continued:
Reagents
Interfering Substances: Amino acids such as citrulline and allantoin interfere
with the assay because they are similar in structure to urea and react with
diacetylmonoxime. The presence of these substances are very low and do
not lead to significant interference.
BUN Color Reagent:
Diacetylmonoxime 1.7 g/L and Thiosemicarbazide 0.3 g/L
BUN Acid Reagent:
Ferric chloride hexahydrate 33.4 mg/dL, Sulfuric acid, Phosphoric acid
Urea Nitrogen Standard (25 mg/dL):
Urea equivalent to 25 mg of BUN/dL in weak sulfuric acid solution.
Urea Nitrogen Standard (50 mg/dL):
Urea equivalent to 50 mg of BUN/dL in weak sulfuric acid solution.
Urea Nitrogen Standard (75 mg/dL):
Urea equivalent to 75 mg of BUN/dL in weak sulfuric acid solution.
Equipment
Procedure
Reagents and standards are ready to use.
Reagents and standards are stable at room temperature until expiration date
on bottle labels.
- Spectrophotometer with absorbance readings at 520 nm
- Pipettes which can accurately deliver 10 uL, 0.5 mL and 1.0 mL
- Pipette bulbs
- Pipette tips
- Water bath at 100 degrees Celsius
- Room temperature water bath
- Stopwatch
- Test tubes
- Vortex mixer
- Level 1 Control
- Level 2 Control
- Sharpies
- Type 1 Water
1. Obtain test tubes and place in portable test tube rack. (At least one
tube each for the blank, 25 standard, 50 standard, 75 standard,
Quality Control 1 and Quality Control 2. At least one tube for each
patient sample.) Label each tube.
2. Pipette into all tubes: 1.0 mL BUN Acid Reagent (Ready to use).
3. Pipette into all tubes: 0.5 mL BUN Color Reagent (Ready to use).
4. Pipette 10 uL water into the blank. Mix well.
5. Pipette 10 uL Urea Nitrogen Standard (25 mg/dL) into standard 25.
Mix well.
Page 2 of 4
CLS Laboratory
San Francisco State University
1600 Holloway Avenue
San Francisco, CA 94132
6. Pipette 10 uL Urea Nitrogen Standard (50 mg/dL) into standard 50.
Mix well.
7. Pipette 10 uL Urea Nitrogen Standard (75 mg/dL) into standard 75.
Mix well.
8. Pipette 10 uL Quality Control 1 into Quality Control tube 1. Mix
well.
9. Pipette 10 uL Quality Control 2 into Quality Control tube 2. Mix
well.
10. Patient blood samples must be centrifuged to separate serum or
plasma. Mild hemolysis will not impact results.
11. Pipette 10 uL of patient plasma or serum into Patient tube. Mix well.
12. Incubate for exactly 10 minutes in a boiling water bath (Water
temperature should be monitored with a thermometer at 100 degrees
Celsius).
13. Remove tubes from boiling water bath and immerse tubes in cold
water bath for 3-5 minutes.
14. Remove tubes from cold water bath. Mix well.
15. Before reading absorbances of tubes, use a Kimwipe to wipe the
outside of the tubes to prevent interference from residues.
16. Read absorbances of all tubes using spectrophotometer at 520 nm.
17. If any tube reads an absorbance error, dilute the sample and measure
the absorbance of the diluted sample. Multiply the new absorbance
by the dilution factor to obtain the absorbance of the undiluted
sample.
18. Construct a standard curve by plotting the absorbance vs.
concentration of the blank and 3 standards. Draw a best fit line.
Determine the Quality Control and patient BUN concentrations using
their absorbances and the best fit line.
19. Construct a data table.
Each run must include a reagent blank and 3 BUN standards. The readings
obtained from the 3 standards are used to construct a standard curve. By
plotting the absorbance readings vs. BUN concentrations (mg/dL) of
standards, a best fit line may be obtained. Using the best fit line and the
absorbances of the quality control and patient samples, concentrations may
be found from the standard curve. Example:
BUN Calibration Curve
0.5
75, 0.437
0.4
Absorbance
Calculations
CLS Laboratory BUN Procedure #900.099
Section: Chemistry
Date of Implementation: June 6, 2015
Date of Revision: Stanbio BUN 0580 retired, April 4, 2015
0.3
50, 0.257
0.2
25, 0.133
0.1
0
0, 0
0
20
40
60
80
Concentration (mg/dL)
Page 3 of 4
CLS Laboratory
San Francisco State University
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory BUN Procedure #900.099
Section: Chemistry
Date of Implementation: June 6, 2015
Date of Revision: Stanbio BUN 0580 retired, April 4, 2015
Calculations continued:
For urine specimens, multiply the observed absorbance by a factor of 20 to
obtain the mg/dL of BUN in the urine sample (since a dilution was
performed – see Specimen Requirements above). To determine the BUN
concentration of a 24 hour urine specimen, use the following calculation:
Interpretation of
Results
BUN (gm/24 hours) = BUN (mg/dL) x Urine Volume (mL)
100,000
Normal Range in Serum/Plasma: 5 – 25 mg/dL
Normal Range in Urine: 6 – 17 gm/24 hours
Normal reference ranges across different laboratories will vary due to
differences in instrumentation and local populations.
Clinical
Applications
Notes
Greatly elevated BUN values may indicate renal disease.
Stanbio Urea Nitrogen (BUN) Procedure # 0580 was designed to measure
urea nitrogen concentrations (mg/dL) in plasma, serum and urine samples.
Plasma and serum should be tested as soon as the blood is drawn and
centrifuged. Urine samples will need Fluoride-Thymol preservative. 24
hour urine samples may also be tested.
- Pipetting microliter quantities of sample and standards are critical to the
accuracy and precision of each run. Use of micropipettes with disposable
tips is strongly recommended. Materials should be pipetted directly to the
bottom of the cuvette or reaction tube.
- All specimens should be thoroughly mixed with reagents before heating.
Vortexing is recommended. Mixing is required after cooling and before
reading absorbances.
References
- Water levels of the boiling water bath must be above the reaction mixture at
all times. The water bath must be large enough to ensure that boiling
continues for the full incubation.
- Stanbio Urea Nitrogen (BUN) Procedure No. 0580
- San Francisco State CLS SOP Template
- San Francisco State CLS Grading Rubric
- Materials Safety Data Sheet (http://www.msds.com/)
Author:____________________________
Date:______________________________
Approved By:_______________________
Date:______________________________
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