Liquid Urea Nitrogen

(BUN)
Reagent Set

Intended Use 3. All material coming in contact with the sample must be free of ammonia and
For the quantitative determination of urea nitrogen in serum. For in vitro heavy metals.7
diagnostic use only. 4. Urea in serum is reported stable for seventy-two hours refrigerated at 2-8°C.
Unrefrigerated sera should be used within eight hours.
Clinical Significance 5. Specimen collection should be carried out in accordance with NCCLS M29-
T2.8 No method can offer complete assurance that human blood samples
Determination of urea nitrogen in serum is widely used as a screening test
will not transmit infection. Therefore, all blood samples should be considered
for renal function. When used in conjunction with the determination of
potentially infectious.
creatinine in serum it is helpful in the differential diagnosis of the three types
of azotemia; pre-renal, renal and post-renal.1 Interferences
1. Urease action is inhibited by fluoride.
Method History 2. Samples with abnormal ammonia levels give falsely elevated BUN results.
Urea has been determined by the direct method2 where urea condenses with 3. Bilirubin to the level of 20 mg/dl was found to exhibit negligible interference
diacetyl to form a chromagen and an indirect method where ammonia is (<10%) in this assay.
measured as a product of urease action on urea.3 The liberated ammonia 4. Hemoglobin to the level of 200 mg/dl was found to exhibit negligible
has been measured using Nessler’s reagent4 and by the Berthelot reaction.5 interference (<5%) in this assay.
Talke and Schubert introduced a totally enzymatic procedure in 1965 utilizing 5. For a comprehensive review of drug interference see Young, et al.9
urease and glutamate dehydrogenase.6 The present procedure is based on
a modification of their method. Materials Provided
Urea Nitrogen Enzyme Reagent (R1).
Principle Urea Nitrogen Coenzyme Reagent (R2).
Urease Materials Required but not Provided
Urea + H2O -------------------------------------------------------------
2 NH3 + CO2
1. Controls
2. Calibrator
GD
3. Beckman Coulter AU™ analyzer
NH3 + α-Ketoglutarate + NADH + H+ ------------
L-glutamate + NAD+ + H20 4. Application and Instrument manuals
Urea is hydrolyzed by urease to produce ammonia and carbon dioxide. The Procedure (Beckman Coulter AU™400 application)
liberated ammonia reacts with α-ketoglutarate in the presence of NADH to
yield glutamate. An equimolar quantity of NADH undergoes oxidation during SPECIFIC TEST PARAMETERS
the reaction resulting in a decrease in absorbance that is directly proportional
TEST NUMBER: # TEST NAME: BUN ∇ TYPE: Serum ∇ OPERATIONAL: Yes ∇
to the urea nitrogen concentration in the sample.
SAMPLE VOL.: 2 DIL. VOL.: 0 PRE-DILUTION RATE: 1
REAGENTS: R1 VOLUME: 150 DIL. VOL.: 0 MIN. OD MAX. OD
Reagent Composition
Working reagent concentrations: Urease (Jack Bean) >15,000 U/L, GLDH R2 VOLUME: 30 DIL. VOL.: 0 L 0.600 H 2.500
(Bovine) >200 U/L, ADP >0.6mM, α-Ketoglutarate 3.6 mM, NADH >0.28 REAGENT OD LIMIT:
mM, Buffer, pH 7.8 ± 0.1, stabilizers, Sodium Azide (0.25%) as preservative. WAVELENGTH: PRI. 340 ∇ SEC. 380 ∇ FIRST L: 0.800 FIRST H: 2.500
METHOD: RATE ∇ LAST L: 0.800 LAST H: 2.500
Reagent Preparation REACTION SLOPE: - ∇ DYNAMIC RANGE:
Reagents provided as ready to use liquids. MEASURING POINT 1: FIRST: 17 LAST: 27 L: # H: #
MEASURING POINT 2: FIRST: LAST: CORRELATION FACTOR:
Reagent Storage and Stability LINEARITY: 25 % A: 1 B: 0
1. Store R1 and R2 reagents at 2-8°C. NO LAG TIME: NO ∇ ON BOARD STABILITY PERIOD: #
2. The R1 and R2 reagents remain stable until expiration date listed on
reagent vial labels when stored tightly capped at 2-8°C. SPECIFIC TEST PARAMETERS

VALUE FLAG: # ∇ LEVEL L: # LEVEL H: #

Precautions
1. This reagent is for in vitro diagnostic use only. NORMAL RANGES: AGE L AGE H
2. Avoid ingestion of reagent as toxicity has not yet been determined. SEX YEAR MONTH YEAR MONTH L H
3. Reagents contain sodium azide (0.25%) as preservative. Sodium azide ○ 1. # ∇ # # # # # #
may react with copper or lead plumbing to form explosive metal azides. ○ 2. # ∇ # # # # # #
Upon disposal flush with large amounts of water. ○ 3. # ∇ # # # # # #
4. All specimens should be handled in accordance with good laboratory
practices using appropriate precautions as described in the CDC/NIH ○ 4. # ∇ # # # # # #
Manual “Biosafety in Microbiological and Biomedical Laboratories,” 2nd ○ 5. # ∇ # # # # # #
ed., 1988, HHS Publication No. (CDC) 88-8395. ○ 6. # ∇ # # # # # #
7. NONE SELECTED # #
Specimen Collection and Storage 8. OUT OF RANGE L H # #
1. Serum is recommended. PANIC VALUE: # # UNIT: mg/dl DECIMAL PLACES: 0
2. Plasma containing anticoagulants should not be used.

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 Liquid Urea Nitrogen
(BUN)
Reagent Set

CALIBRATION SPECIFIC PARAMETERS Expected Values

7-18 mg/dl7
CAL TYPE: AB ∇ FORMULA : Y=AX+B ∇ COUNTS: 2 PROCESS: CONC. ∇ It is strongly recommended that each laboratory establish its own reference range.
CAL. NO. OD CONC. FAC/OD-L FAC/OD-H Performance
POINT 1. # # -9999999 9999999 1. Linear Range: 0-150 mg/dl. Samples that exceed 150 mg/dl should be
POINT 2. diluted with an equal volume of saline and re-assayed. Multiply the result by
POINT 3. two.
POINT 4. 2. Analytical Measuring Range: 0-150 mg/dl
POINT 5. 3. Comparison: A comparison study performed between the Beckman Coulter
POINT 6. AU400 and Roche Hitachi 717 using this method resulted in a correlation
POINT 7. coefficient of r = 0.995 and the regression equation of y = 1.075x – 0.16.
(n = 38, range 8 – 45 mg/dl)
1-POINT CAL. POINT: ○ WITH CONC-0
4. Precision:
MB TYPE FACTOR: CALIBRATION STABILITY PERIOD: #
Within - day precision study was performed using three levels of material.
#: User-Defined Between - day precision study was performed using two levels of control
The above reagent parameters are intended to serve as a guide for use with material assayed over a 20 day period with 2 runs per day and 2 replicates
Pointe Scientific, Inc. reagent. The parameters are based on data generated per run.
by Pointe Scientific, Inc. Please note: These parameters should be used in Within Day (N=20) Day to Day
conjunction with your laboratory Quality Control Program for validation. Mean S.D. C.V.% Mean S.D. C.V.%
5.9 0.3 5.1 16.3 0.5 2.9
NOTE: For other instrument specific applications please contact Pointe 17.5 0.6 3.4 55.1 1.2 2.1
Scientific, Inc. Technical Service Department at 1-800-445-9853 40.7 0.9 2.2
Limitations Precision and Linearity studies were performed following modifications of
Samples with values above 150 mg/dl should be diluted with 0.9% saline 1:1, CLSI Protocols EP5 and EP610 using a Beckman Coulter AU™400 analyzer
re-assayed and the results multiplied by two.
5. Sensitivity: The sensitivity for the Liquid BUN reagent was investigated by
Calibration reading the change in absorbance at 340 / 380 nm for a saline sample, and
The procedure should be calibrated according to the instrument serum samples with known concentrations. Ten replicates of each sample
manufacturer’s calibration instructions. If control results are found to be out were performed. The results of this investigation indicated that, on the
of range, the procedure should be recalibrated. analyzer used, the Liquid BUN reagent showed little or no drift on a zero
sample. Under the reaction conditions described, 1 mg/dl of BUN gives an
Calculation absorbance of 0.001.
(A1-A2) = Absorbance change between readings
References
(A1 – A2) unknown x concentration = BUN (mg/dl) 1. Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia W.B.
(A1 – A2) standard of standard Saunders (1976).
2. Fearon, W.R., Biochem J. 331:902 (1939).
Example: If the unknown had an A1 = 1.5 and A2 = 1.0, the standard A1 = 3. Marshall, E.K., Jr., J. Biol. Chem. 15:487 (1913).
1.5 and A2 = 0.9 and the concentration of the standard = 20 mg/dl then: 4. Gentzkow, C.J., J. Biol. Chem. 143:531 (1952).
5. Fawcett, J.K., Scott, J.E., J. Clin. Path. 13:156 (1960).
(1.5 – 1.0) = 0.5 x 20 = 17 mg/dl 6. Talke, H., Schubert, G.E., Klin. Wschr. 43:174 (1965).
(1.5 – 0.9) 0.6 7. Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia W.B.
NOTE: To obtain results in SI units multiply by 10 to convert dl to liters Saunders, p. 991 (1976).
and divide by 28, the molecular weight of nitrogen. 8. NCCLS document “Protection of Laboratory Workers from Infectious Disease
Transmitted by Blood, Body Fluids, and Tissue”, 2nd Ed. (1991).
Example: 17 mg/dl x 10/28 = 6.06 mmol/L. 9. Young, D.S., et al, Clin. Chem. 21:1D (1975).
10. CLSI protocols EP5-A2, Vol. 24 No. 25, 2nd Ed, (2004) and CLSI EP6-A, Vol.
To convert mg/dl Urea Nitrogen to mmol Urea/L, multiply the mg/dl Urea
23 No. 16 (2003)
Nitrogen value by 0.357.
To convert mg/dl Urea Nitrogen to mg/dl Urea, multiply the mg/dl Urea
Nitrogen value by 2.14. Manufactured by Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188
Quality Control
The validity of the reaction should be monitored by use of the control sera European Authorized Representative:
with known normal and abnormal BUN values. These controls should be run Obelis s.a.
at least with every working shift in which urea nitrogen assays are performed. Boulevard Général Wahis 53
It is recommended that each laboratory establish its own frequency of control 1030 Brussels, BELGIUM
determination. Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: mail@obelis.net

Rev. 1/13 P803-OB752-01