CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 1 of 6
STANBIO UREA NITROGEN (BUN)
Quantitative Determination of Urea Nitrogen in Serum, Plasma, or Urine
Policy
The laboratory operates in strict accordance with CLSI GP2-A5 guidelines
by identifying laboratory procedures using work processes in the
laboratory’s operational path of workflow and writes procedures for preanalytical, analytical, and post-analytical laboratory practices.
Purpose
The STANBIO BUN method employs the diacetylmonoxime (DAM)
methodology. In this condensation reaction, urea directly reacts with DAM
under acid conditions. In the presence of thiosemicarbazide, a red-purple
chromogen forms and is measured spectrophotometrically at a wavelength
of 520nm.
Reaction Sequence:
Thiosemicarbazide
+
Urea
Responsibility
Red-Purple Chromogen
Diacetylmonoxime
The CLS Internship Program Director shall review and approve procedural
documents, appoint personnel for document implementation, control, and
record.
Interns, faculty, and any other individuals operating within the San Francisco
State University CLS Laboratory must refer to this SOP and operate within
its limits when performing the STANBIO BUN procedure.
Clinical Utility
The major pathway of nitrogen excretion is in the form of urea that is
synthesized in the liver, released into the blood, and cleared by the kidneys.
A high serum urea nitrogen occurs in glomerulonephritis, shock, urinary
obstruction, pyelonephritis, and other causes of acute and chronic renal
failure. Severe congestive heart failure, protein hyperalimentation, diabetic
ketoacidosis, dehydration, and bleeding from the gastrointestinal tract
elevate BUN. Low BUN often occurs in normal pregnancy, decreased
protein intake, in acute liver failure, and with intravenous fluid
administration.1
CPT Code
12345-0580
Specimen
Requirements
Specimens recommended for testing are serum, plasma, or, urine.
Page 1 of 6
CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 2 of 6
Serum and plasma urea nitrogren is stable for 1 day at room temperature
(15-25°C); several days refrigerated (2-8°C); and 6 months when frozen
at -20°C.
Serum: Hemolysis can alter results; therefore, serum specimens should be
analyzed as soon as possible.
Plasma: Plasma specimens may be used as long as anticoagulant used
does not contain ammonium salts.
Urine: Urine Specimens should be preserved with Fluoride-Thymol. Add
19.0 mL of water to 1.0 mL of diluted urine for analysis and multiply the
observed value by a factor of 20 to obtain the mg/dL of BUN in the urine
specimen.
For 24 hour urine specimen:
gram
)
24hours
BUN (
Materials
= (BUN (
mg
) x (Urine
dL
Equipment
 Pipettes
o To accurately deliver:
 20uL
 2.0mL
 1.0mL
 Test tubes
 Vortex Mixer
 Heat block or water bath, 100°C
 Cold Water Bath


Spectrophotometer with 520nm
absorbance reading capability
Timer
Volume (mL)))/100,000
Reagents
 BUN Color Reagent, Catalog No.
0584
o Diacetylmonoxime
1.7g/L
o Thiosemicarbazide 0.3g/L
 BUN Acid Reagent, Catalog No.
0585
o Ferric Chloride Hexahydrate
33.4mg/L
o Sulfuric Acid
o Phosphoric Acid
 Urea Nitrogen Standard,
(25mg/dL) Catalog No. 0581
o Contains Urea equivalent to
25 mg of BUN/dL in a weak
sulfuric acid solution
 Urea Nitrogen Standard,
(50mg/dL) Catalog No. 0582
o Contains Urea equivalent to
50 mg of BUN/dL in a weak
sulfuric acid solution
 Urea Nitrogen Standard,
(75mg/dL) Catalog No. 0583
o Contains Urea equivalent to
75 mg of BUN/dL in a weak
sulfuric acid solution
Page 2 of 6
CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
Procedure
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 3 of 6
Step Action
1
Pipette the following volumes (in milliliters) to the corresponding
test tube:
Reagent
Standard
Sample (U)
Blank (B)
(S)
Sample
0.02
Standard
0.02
Water
0.02
BUN Color
Reagent
1.0
1.0
1.0
BUN Acid Reagent
2.0
2.0
2.0
*Mix well with each addition.
*There will be three standard tubes – one for each standard:
25mg/dL, 50mg/dL, and 75 mg/dL.
2
Mix the test tubes and incubate for exactly 10 minutes in a boiling
water bath. If a heat block is used, incubate for 12 minutes at
100°C.
Remove the tubes from water bath/heat block simultaneously and
immerse in cold water for 3-5 minutes.
Remove from cold water bath, mix well and read absorbance (A)
of unknown and standard against Reagent Blank at 520nm.
3
4
*Notes:
 Color is stable for 30 minutes.
 A reagent blank and 3 BUN standards must be run with every test.



The readings obtained with the 3 standards are used to prepare a
standard curve for each run.
The reaction departs from linearity at levels below about 3 mg/dL,
suggesting that it does not strictly follow the Beer-Lambert Law.
Pipetting proper volumes of reagents and specimens is crucial.
Use of micropipettes is recommended.
Mixing of all reaction tubes with each addition of
reagent/specimen as well as before and after heating is
Page 3 of 6
CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 4 of 6

essential. “Vortexing” is recommended.
During heating, ensure that water levels remain above reaction
mixtures and that heating time is adequate according to
method employed. If a heat block is used, heating time may be
slightly extended.
Interpretation
of Results
Calculations
A reagent blank and 3 BUN standards must be run with every test. The
readings obtained with the 3 standards are used to prepare a standard
curve for each run. Derive BUN concentration of specimens using obtained
absorbance.
If using urine specimen, refer to “Specimen Requirements” section for
calculation of BUN as well as urine specimen calculation.
Expected
Results
The provided ranges should be used as a guideline.
Serum/Plasma
Urine
Clinical
Significance
5-25 mg/dL
6-17 grams/24 hrs
BUN determination is useful in the evaluation of cardiac and renal function
and may also yield information for other health conditions.
Plasma BUN levels of 37 mg/dL or above are considered significantly high.
See also section “Clinical Utility.”
Interference
Substances similiar to urea and which react with diacetylmonoxime in an
analogous manner are the amino acids citrulline and allantoin. The presence
of these materials are very low and do not lead to significant interference.
The effects of drugs and their metabolites should be considered.
Method
Performance
Specification
Specific for BUN given the absence of citrulline and allantoin.
Between Run Precision: +/- 5%
Recoveries: 92-99%
Reporting
Report results after confirmation that controls are valid and no errors were
detected.
Page 4 of 6
CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 5 of 6
Supporting
Documents
Related
Procedures

Pointe 180 Analyzer – Urea Nitrogen (BUN) Reagent Set
References
1. Tietz NW (ed). Fundamentals of Clinical Chemistry. Ed. 3,
Philadelphia: WB Saunders; 967; 1987.
2. Stanbio Laboratory. Stanbio Urea Nitrogen (BUN) Procedure No.
0580. Boerne, Texas: 2005.
MSDS:
DAM:
doc#146078
Ferric Chloride Hexahydrate: doc# 1982833
Phosphoric Acid:
doc#1855291
Sulfuric Acid:
doc#2360643
Thiosemicarbazide:
doc#1778034
Urea:
doc#2801379
Author:_________________
Date:__________________
Approved by:______________
Date:____________________
Page 5 of 6
CLS Laboratory
San Francisco State U niversity
1600 Holloway Avenue
San Francisco, CA 94132
CLS Laboratory Procedure #STANBIO-BUN-0580
Section: Chemistry
Date of Implementation: 11/29/2010
Page 6 of 6
Procedural Flow Chart: Stanbio Urea Nitrogen (BUN)
I. PREANALYTICAL
Specimen is urine, serum, or
plasma?
Yes - Was the stored properly
handled: storage, temperature,
no hemolysis, proper
anticoagulant/additive used,
etc?
II. ANALYTICAL
(To be done with each
specimen)
Reagent Blank (B): In a test
tube, add 0.02 mL of water, 1.0
mL BUN Color Reagent, and 2.0
mL BUN Acid Reagent. Mix.
In a test tube, add 0.02 mL of
specimen, 1.0 mL BUN Color
Reagent, and 2.0 mL BUN Acid
Reagent. Mix.
III. POSTANALYTICAL
STANDARD: In a test tube add,
0.02 mL of standard, 1.0 mL BUN
Color Reagent, and 2.0 mL BUN
Acid Reagent. Mix.
Use standard absorbances to
construct a standard curve
. X-axis: Concentration (mg/dL)
(Repeat for remaining
standards).
Y-axis: Absorbance(A) at 520 nm
Incubate for exactly 10 minutes
in a boiling water bath. If a heat
block is used, incubate for 12
minutes at 100°C.
Incubate for exactly 10 minutes
in a boiling water bath. If a heat
block is used, incubate for 12
minutes at 100°C.
Incubate for exactly 10 minutes
in a boiling water bath. If a heat
block is used, incubate for 12
minutes at 100°C.
From the standard curve, derive
the value(s) of specimen(s).
Yes: Proceed to Analytical Phase
Remove the tubes from water
bath/heat block simultaneously
and immerse in cold water for 35 minutes.
Remove the tubes from water
bath/heat block simultaneously
and immerse in cold water for 35 minutes.
Remove the tubes from water
bath/heat block simultaneously
and immerse in cold water for 35 minutes.
Confirm that controls/standards
are valid and no errors are
detected.
No: Request new aliquot or
specimen collection
Remove from cold water bath,
mix well and read absorbance
(A) of Reagent Blank at 520nm.
Remove from cold water bath,
mix well and read absorbance
(A) of specimen against Reagent
Blank at 520nm.
Remove from cold water bath,
mix well and read absorbance
(A) of standard against Reagent
Blank at 520nm.
Yes: Report results.
Record Reagent Blank (B)
absorbance.
Record specimen absorbance.
Record specimen absorbance.
No: Troubleshoot errors.
No - Request proper specimen
type.
Proceed to Postanalytical Phase.
Proceed to Postanalytical Phase.
Proceed to Postanalytical Phase.
Page 6 of 6