HORIBA Instruments Incorporated

5449 Research Drive, Canton, MI 48188

Pointe Phone: 734-487-8300; (800) 445-9853
Urea Nitrogen (BUN)
Reagent Set
Intended Use
For the quantitative determination of urea nitrogen in serum using the Yumizen C560 analyzer. Rx Only.

Clinical Significance
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; pre-renal, renal and post-renal.1

Method History
Urea has been determined by the direct method2 where urea condenses with diacetyl to form a chromagen and an indirect method where ammonia is measured as a
product of urease action on urea.3 The liberated ammonia has been measured using Nessler’s reagent4 and by the Berthelot reaction.5 Talke and Schubert
introduced a totally enzymatic procedure in 1965 utilizing urease and glutamate dehydrogenase.6 The present procedure is based on a modification of their method.

Principle
Urease
Urea + H2O 2 NH3 + CO2
GD
NH3 + -Ketoglutarate + NADH + H+ L-glutamate + NAD+ + H20
Urea is hydrolyzed by urease to produce ammonia and carbon dioxide. The liberated ammonia reacts with -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
nitrogen concentration in the sample.

Reagent Composition
Working reagent concentrations: Urease (Jack Bean) >15,000 U/L, GLDH (Bovine) >200 U/L, ADP >0.6 mM, -Ketoglutarate 3.4 mM, NADH >0.28 mM, Buffer, pH
7.8 ± 0.1, stabilizers, Sodium Azide (0.28%) as preservative.

Reagent Preparation
The reagents are ready to use.

Reagent Storage
Store R1 and R2 reagents at 2-8°C. The reagents are stable until the expiration date appearing on the label when stored as directed. Manufacturer studies have
shown reagent is stable for 30 days once placed in the refrigerated reagent carousel (2-10°C), however reagent stability may vary based on individual laboratory
conditions.

Reagent Deterioration
The reagent should not be used if the working reagent has a reagent blank absorbance less than 1.0 at 340 nm.

Precautions and Hazards

1. This reagent is for in vitro diagnostic use only.
2. Avoid ingestion of reagent as toxicity has not yet been determined.
3. Reagents contain sodium azide (0.28%) as preservative. Sodium azide may react with copper or lead plumbing to form explosive metal azides. Upon disposal
flush with large amounts of water.
4. All specimens should be handled in accordance with good laboratory practices using appropriate precautions as described in the CDC/NIH Manual “Biosafety in
Microbiological and Biomedical Laboratories,” 2nd ed., 1988, HHS Publication No. (CDC) 88-8395.
Hazards:
R1: Hazard Classifications: Specific Target Organ Toxicity, Single Exposure; Respiratory System (Category 3)
Hazard Statement: H335: May cause respiratory irritation.
Precautionary Statements: Prevention: P261 Avoid breathing dust/fume/gas/mist/vapors/spray. P271 Use only in a well-ventilated area. Response: P312
Call a POISON CENTER or doctor/physician if you feel unwell. P304 + P340 IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for
breathing. Storage: P403 + P233 Store in a well-ventilated place. Keep container tightly closed. Disposal: P501: Dispose of contents into sewer system after
diluting with large volumes of water, if in accordance with local regulations. Signal Word: Warning
R2: Hazard Classifications: Acute Toxicity, Dermal (Category 4)
Hazard Statement: H312: Harmful in contact with skin.
Precautionary Statements: Prevention: P280 Wear protective gloves/protective clothing/eye protection/face protection. Response: P312 Call a POISON CENTER or
doctor/physician if you feel unwell. P363 Wash contaminated clothing before reuse. P302 + P352 IF ON SKIN: wash with plenty of soap and water. Disposal: P501:
Dispose of contents into sewer system after diluting with large volumes of water, if in accordance with local regulations. Refer to the Safety Data Sheet for this
product (SDS-B7552) available by calling 1-734-487-8300.
Signal Word: Warning
Specimen Collection and Storage
1. Serum is recommended.
2. Plasma containing anticoagulants should not be used.
3. All material coming in contact with the sample must be free of ammonia and heavy metals.7
4. Urea in serum is reported stable for seventy-two hours refrigerated at 2-8°C. Unrefrigerated sera should be used within eight hours.
5. Specimen collection should be carried out in accordance with NCCLS M29-T2.8 No method can offer complete assurance that human blood samples will not
transmit infection. Therefore, all blood samples should be considered potentially infectious.
 Pointe
Urea Nitrogen (BUN)
Reagent Set

Interferences
1. Urease action is inhibited by fluoride.
2. Samples with abnormal ammonia levels give falsely elevated BUN results.
3. Bilirubin to the level of 20 mg/dl was found to exhibit negligible interference (<2%) in this assay.
4. Hemoglobin to the level of 200 mg/dl was found to exhibit negligible interference (<5%) in this assay.
NOTE: The BUN level was 46.0 mg/dl for the Bilirubin study and 46.3 mg/dl for the Hemoglobin study.
5. For a comprehensive review of drug interference see Young, et al.9
Materials Provided
Urea Nitrogen Enzyme Reagent (R1), Urea Nitrogen Coenzyme Reagent (R2)
Materials Required but not Provided
1. Yumizen C560 Analyzer
2. Yumizen C560 Operation manual
3. Chemistry Calibrator, catalog number C7506-50
4. Chemistry control, catalog number C7592-100
Limitations
Samples with values above 150 mg/dl should be diluted with 0.9% saline 1:1, re-assayed and the results multiplied by two.
Calibration
Use Pointe Chemistry Calibrator (Catalog Number: C7506-50). The procedure should be calibrated according to the instrument manufacturer’s calibration
instructions. If control results are found to be out of range, the test may need to be re-calibrated. Under typical operating conditions manufacturer calibration stability
studies have shown the calibration curve will be stable for at least 14 days.
Quality Control
The validity of the reaction should be monitored by use of the control sera with known normal and abnormal BUN values. These controls should be run at least with
every working shift in which urea nitrogen assays are performed. It is recommended that each laboratory establish its own frequency of control determination. Quality
control requirements should be performed in conformance with local, state, and/or Federal regulations or accreditation requirements.
Expected Values
7-18 mg/dl7
It is strongly recommended that each laboratory establish its own reference range.
Performance
1. Assay Range: 1-150 mg/dl. Samples that exceed 150 mg/dl should be diluted with an equal volume of saline and re-assayed. Multiply the result by two.
2. Comparison: A study was performed between the Yumizen C560 and a similar analyzer using this method resulting in the following:
Method BUN
N 84
Mean BUN (mg/dL) 27.7
Range (mg/dL) 5-149
Standard Deviation 29.0
Regression Analysis y = 0.994x – 0.7
Correlation Coefficient 0.9962
3. Precision: Precision studies were performed using the Yumizen C560 analyzer following a modification of the guidelines which are contained in NCCLS
document EP5-T2.10
Within Day Total
Sample LOW MID HIGH Sample LOW MID HIGH
N 20 20 20 N 40 40 40
Mean 12.7 47.4 131.9 Mean 13.0 48.8 134.3
Standard Deviation 0.7 0.7 0.9 Standard Deviation 0.8 1.7 2.5
Coefficient of Variation (%) 5.2% 1.4% 0.6% Coefficient of Variation (%) 7.2% 2.5% 1.9%
4. Sensitivity: 2SD limit of detection (95% Conf) = 1 mg/dL
References
1. Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia W.B. Saunders (1976).
2. Fearon, W.R., Biochem J. 331:902 (1939).
3. Marshall, E.K., Jr., J. Biol. Chem. 15:487 (1913).
4. Gentzkow, C.J., J. Biol. Chem. 143:531 (1952).
5. Fawcett, J.K., Scott, J.E., J. Clin. Path. 13:156 (1960).
6. Talke, H., Schubert, G.E., Klin. Wschr. 43:174 (1965).
7. Tietz, N.W., Fundamentals of Clinical Chemistry, Philadelphia W.B. Saunders, p991 (1976).
8. NCCLS document “Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids, and Tissue”, 2 nd Ed. (1991).
9. Young, D.S., et al, Clin. Chem. 21:1D (1975).
10. NCCLS document “Evaluation of Precision Performance of Clinical Chemistry Devices”, 2nd Ed. (1992).
 HORIBA Instruments Incorporated
5449 Research Drive, Canton, MI 48188
Pointe Phone: 734-487-8300; (800) 445-9853
Urea Nitrogen (BUN)
Reagent Set
CHEMISTRY PARAMETERS

Chem: Urea Nitro No.: 206 Sample Type: Serum

Chemistry: Urea Nitrogen (Liquid) Print Name: Urea Nitro
Reaction Type: Fixed Time Reaction Direction: Negative
Pri Wave: 340 Sec Wave: 660
Unit: mg/dL Decimal 0
Blank Time: 47 49 Reaction Time: 55 63

Sample Vol. Aspirated Diluent Reagent Vol. Diluent

Standard: 1.5 ul --- ul --- ul R1: 150 ul --- ul
Decreased: --- ul --- ul --- ul R2: --- ul -- ul
Increased: --- ul --- ul --- ul R3: --- ul -- ul
 Sample Blank  Auto Rerun R4: --- ul --- ul

Slope/Offset Adjustment
Slope: 1 Offset: 0

Linearity Range (Standard) 1 150 Linearity Limit:

Linearity Range (Decreased) ____ ____ Substrate Depletion:

Linearity Range (Increased) ____ ____ Mixed Blank Abs:

R1 Blank Abs: ____ ____ Uncapping Time

Blank Response: ____ ____ Reagent Alarm Limit:

Twin Chemistry:  Enzyme Linear Extension

 Prozone Check ○Rate Check ○ Antigen Addition

Q1: Q2: Q3: Q4:

PC: ABS:
 Pointe
Urea Nitrogen (BUN)
Reagent Set

CALIBRATION PARAMETERS

Calibrator Definition
Calibrator: * Lot No.: *
Exp Date: *

Carousel Pos
Sample Carousel 1 *
Sample Carousel 2
Sample Carousel 3
Reagent/Calibration

Calibrator Pos Lot No Exp Date Chem Conc Unit

Water W * * BUN 0 mg/dL
Chemistry Calibrator * * * BUN * mg/dL

Calibration Setup
Chem: Urea Nitro

Calibration Settings
Math Model: Two-Point Linear
Factor: Replicates: 2

Acceptance Limits
Cal Time: 336 Hour
Slope Diff: --- SD: ---
Sensitivity : --- Repeatability: ---
Deter Coeff: ---

Auto Calib.
 Bottle Changed  Lot Changed  Cal Time

It is recommended that two levels of control material be assayed daily.

Certified to Perform Reagents
The Pointe reagents are certified to be
* Indicates user defined parameter. manufactured according to specified
14-B7552-600 Manufactured by HORIBA Instruments Inc.- Pointe Brand parameters. Any Pointe reagent product
5449 Research Drive Canton, MI 48188 not meeting specifications through its
listed expiration date will be remedied
immediately without charge.
Manufactured by HORIBA Instruments Incorporated – Pointe Brand
5449 Research Drive, Canton, MI 48188 Symbol Key

European Authorized Representative: Use by (YYYY-MM-DD) Lot and batch code Catalog number
Obelis s.a.
Manufacturer Temperature limitation Consult instructions for use
Boulevard Général Wahis 53
1030 Brussels, BELGIUM In vitro diagnostic medical device Rx Only: Prescription Use Only
Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: mail@obelis.net
Rev. 11/23 P803-B7552-560