Procedure:
CREATININE
OSR6178 and OSR6678
This procedure is valid for the following chemistry analyzers:
· AU400/AU400e / · AU640/AU640e· AU480 / · AU680
· AU600 / · AU2700
· AU5400 / · AU5800
Prepared By / Date Adopted / Supersedes Procedure #
Review Date / Revision Date / Signature
Distributed to / # of
Copies / Distributed to / # of
Copies
PRINCIPLE:
Measurements of creatinine are used in the diagnosis and treatment of renal disease. Serum creatinine measurements prove useful in evaluation of kidney glomerular function and in monitoring renal dialysis. However, the serum level is not sensitive to early renal damage and responds more slowly than blood urea nitrogen (BUN) to hemodialysis during treatment of renal failure. Both serum creatinine and BUN are used to differentiate prerenal and postrenal (obstructive) azotemia. An increase in serum BUN without concomitant increase of serum creatinine is key to identifying prerenal azotemia. With postrenal azotemia, both serum BUN and creatinine rise, but the rise is disproportionately greater for BUN.1
Serum creatinine varies with the subject’s age, body weight, and sex. It is sometimes low in subjects with relatively small muscle mass, cachetic patients, amputees, and in older persons. A serum creatinine level that would usually be considered normal does not rule out the presence of impaired renal function.
INTENDED USE:
System reagent for the quantitative determination of Creatinine in human serum, plasma, or urine on Beckman Coulter AU Clinical Chemistry analyzers.
Creatinine reagent OSR6678 for use on the AU680, AU2700 and AU5400 analyzers only.
METHODOLOGY:
The Beckman Coulter AU System Creatinine procedure is a kinetic modification of the Jaffe procedure2 in which creatinine reacts with picric acid at alkaline pH to form a yellow-orange complex. However, this reaction is not completely specific for creatinine since other reducing substances such as glucose, pyruvate, ascorbic acid, and acetoacetates will react with picrate to form a similar color.3 Fabiny and Ertingshausen4 found that alkaline creatinine picrate reaches maximum color development at a different rate than pseudo-creatinine material. Cook5 utilized different reaction rates of alkaline picrate positive substances to obtain greater specificity with the Jaffe reaction.
The rate of change in absorbance at 520/800nm is proportional to the creatinine concentration in the sample.
Creatinine + Alkaline Picrate ¾¾¾ Yellow-Orange Complex
SPECIMEN:
Patient Preparation:
None required.
Additional instructions for patient preparation as designated by this laboratory:Type:
Serum or heparinized samples free from hemolysis are the recommended specimens and should be separated from the red cells as soon as possible.
Urine specimens should be collected into a clean, leak-proof container. If urine must be collected with a preservative for other analytes, 6N HCL or Boric Acid can be used.6
Additional type conditions as designated by this laboratory:Handling Conditions:
Serum specimens are stable for 7 days refrigerated at 2 - 8°C and indefinitely when frozen (-20°C).8 Creatinine in urine is stable for 2 days at room temperature (15-25°C) and for 6 days refrigerated at 2 - 8°C.7
Additional handling conditions as designated by this laboratory:EQUIPMENT AND MATERIALS:
Equipment:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, AU5400 and AU5800 analyzers.
Materials:
Beckman Coulter AU System Creatinine Reagent
Final concentration of reactive ingredients:
Sodium hydroxide / 120 mmol/LPicric acid / 2.9 mmol/L
Also contains preservatives.
Reagent storage location in this laboratory:Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).
Storage location of test tubes or sample cups in this laboratory:Beckman Coulter Chemistry Calibrator (Cat. No. DR0070)
Beckman Coulter Urine Chemistry Calibrator (Cat. No. DR0091)
Storage location of the calibrator in this laboratory:Precautions:
1. For in vitro diagnostic use.
2. R1 is corrosive and causes burns. Do not pipette by mouth. Avoid contact with eyes, skin or clothing. In case of contact, immediately rinse affected area with plenty of water for 15 minutes. Obtain medical attention immediately for eye contact or ingestion. Do not induce vomiting.
3. R2 is POISON. Do not pipette by mouth. In case of external contact, immediately flush affected area with plenty of water for 15 minutes. Obtain medical attention immediately for eye contact or ingestion.
4. R2 contains picric acid. Dry picric acid explodes when rapidly heated or subjected to percussion. Dilute any spills with water and wipe up immediately.
5. WARNING: POTENTIAL BIOHAZARDOUS MATERIAL. The calibrator is manufactured from human serum. No test method can offer complete assurance that HIV- 1/2, HCV, Hepatitis B, or other infectious agents are absent from biological materials, all calibrator material should be handled at the Biosafety Level 2 as recommended for any infectious human serum or blood specimen in the CDC/National Institutes of Health manual, Biosafety in Microbiological and Biomedical Laboratories, 1993.
6. Do not ingest – Toxicity of DR0091 has not been established
Preparation:
The Beckman Coulter AU System Creatinine Reagent is liquid, ready for use. No preparation is needed.
For OSR6678, insert the pipe supplied into the 180mL Creatinine reagent bottles before use on the analyzer. Care must be taken when handling the pipe to avoid contamination. The pipe is for single use only. Do not remove the large cap on the 180mL bottles.
The Beckman Coulter Chemistry Calibrator reconstitution:
· Remove the vials of calibrator and diluent from storage and let stand at room temperature (15-25°C) for 5 minutes.
· Remove the cap and stopper from the vials of the lyophilized serum and reconstituting diluent.
· Using a volumetric pipette or a calibrated air-displacement pipettor, add exactly 5.0 mL of reconstituting diluent to DR0070 lyophilized serum vial. DO NOT pour directly from the reconstituting diluent vial.
· Replace the cap and stopper to the vial of lyophilized serum immediately after adding the diluent
· Allow the calibrator to stand for 5-10 minutes. Gently swirl the contents until completely dissolved.
The Beckman Coulter Urine Creatinine Calibrator is liquid, ready for use. No preparation is needed.
Storage Requirements:
1. The unopened reagents are stable until the expiration date printed on the label when stored at 2 - 8°C. R1 is light sensitive; store in the dark before placing on the instrument.
2. Opened reagents are stable for 7 days when stored in the refrigerated compartment of the analyzer.
3. Contamination after opening must be avoided.
4. Un-reconstituted calibrator and diluent are stable until the expiration date stated on the label when stored at 2 - 8°C.
5. For Creatinine, reconstituted calibrator materials are stable for 7 days from the date of reconstitution when stored at 2 - 8°C. The materials should be capped and stored upright 2 - 8°C when not in use.
6. Unopened bottles of DR0091 Urine Creatinine Calibrator are stable until the expiration date printed on the label. Store at 2 - 8°C.
7. Opened bottles of DR0091 Urine Creatinine Calibrator are stable for 8 months. Store at 2 - 8°C.
Indications of Deterioration:
Discoloration of the reagent, visible signs of microbial growth, turbidity or precipitation in reagent may indicate degradation and warrant discontinuance of use.
Additional storage requirements as designated by this laboratory:PERFORMANCE PARAMETERS:
The following data was obtained using this Creatinine Reagent on Beckman Coulter AU analyzers according to established procedures. Results obtained at individual facilities may differ.
Precision:14
Estimates of precision, based on CLSI recommendations12, are consistent with typical performance. The within run precision for serum samples is less than 3%CV and total precision is less than 6%CV. Assays of control sera were carried out and the data reduced following CLSI guidelines.
SERUMN=100 / Within run / Total
Mean, mg/dL / SD / CV% / SD / CV%
1.00 / 0.01 / 1.0 / 0.01 / 1.0
5.60 / 0.05 / 0.8 / 0.09 / 1.5
URINE
N=100 / Within run / Total
Mean, mg/dL / SD / CV% / SD / CV%
96.5 / 0.60 / 0.6 / 1.00 / 1.0
195.9 / 1.10 / 0.5 / 2.50 / 1.3
Method Comparison:14
Patient samples were used to compare this Creatinine Reagent. Representative performance data on AU analyzers is shown in the next table.
SERUMY Method / AU640/ AU640e
X Method / AU600
Slope / 1.029
Intercept / -0.01
Correlation Coeff. (r) / 0.9996
No. of Samples (n) / 175
Range (mg/dL) / 0.2 – 16.0
URINE
Y Method / AU640/ AU640e
X Method / AU600
Slope / 1.000
Intercept / 0.9
Correlation Coeff. (r) / 0.9998
No. of Samples (n) / 176
Range (mg/dL) / 11 - 281
Sensitivity:
Typical change in absorbance for 1 mg/dL of Creatinine is 12.5 mAbsorbance.
CALIBRATION:
Standard Preparation:
For Serum: Perform a two-point calibration (AA) using a water blank (blue rack) and the appropriate calibrator in a yellow calibration rack. The frequency of calibration is daily. Calibration of this creatinine procedure for serum determinations is accomplished by use of the Beckman Coulter Chemistry Calibrator (Cat No. DR0070), which is traceable to an isotope dilution mass spectrometry (IDMS) reference method using the National Institutes of Standards and Technology (NIST) Standard Reference Material 967.
For Urine: Perform a one-point calibration (AB) using a water blank (blue rack) and the appropriate calibrator in a yellow calibration rack. The frequency of calibration is daily. Calibration of this creatinine procedure for urine determinations is accomplished by use of the Beckman Coulter Urine Chemistry Calibrator (Cat No. DR0091). The assay value for creatinine is traceable to the National Institute of Standards and Technology (NIST)
Calibration Procedure:
Calibrate the reagent on a daily basis and run QC at a minimum of every eight (8) hours as part of your laboratory’s quality control program. Recalibrate the reagent when there is a drift in QC recovery outside of your laboratory’s acceptance limits.
Recalibration of this test is required when any of these conditions exist:
1. A reagent lot number has changed or there is an observed shift in control values.
2. A fresh bottle of reagent is used for testing.
3. Major preventative maintenance was performed on the analyzer or critical part was replaced.
Absorption of atmospheric CO2 by the reagent on board the analyzer can impair calibration stability. This effect will vary depending upon the rate of use.
QUALITY CONTROL:
During operation of the Beckman Coulter AU analyzer at least two levels of an appropriate quality control material should be tested. Run QC at a minimum of every eight (8) hours. Recalibrate the reagent when there is a drift in QC recovery outside your laboratory’s acceptance limits.
In addition, controls should be performed after calibration, with each new lot of reagents, and after specific maintenance or troubleshooting steps described in the appropriate AU User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and each laboratory’s standard procedure. Appropriate qualified urine controls should be established and utilized during urine analysis. The effect of the atmospheric CO2 uptake by the reagent on board the analyzer is more pronounced as the volume in the reagent vial is reduced. Control frequency should be adjusted accordingly to demonstrate continued calibration stability.
Location of controls used at this laboratory.ANALYZER PARAMETERS:
A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at www.beckmancoulter.com.
CALCULATIONS:
For SI units (mmol/L), multiply the results by 88.4.
REPORTING RESULTS:
Reference Ranges:
Serum11 / Urine1Male: / 0.7 - 1.3 mg/dL / 14 - 26 mg/kg/24 hours
Female: / 0.6 - 1.2 mg/dL / 11 - 20 mg/kg/24 hours
Beckman Coulter Determined Range13: / 0.6 – 1.3 mg/dL
Expected values may vary with age, sex, diet and geographical location. Each laboratory should determine its own expected values as dictated by good laboratory practice.
Expected reference ranges in this laboratory:Procedures for Abnormal Results:
Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.
Reporting Format:
Results are automatically printed for each sample in mg/dL at 37°C.
Additional reporting information as designated by this laboratory:LIMITATIONS:
The Beckman Coulter AU System Creatinine procedure is linear from 0.2 up to 25.0 mg/dL for serum determinations and from 1 - 300 mg/dL for urine determinations. Samples exceeding the upper limit of linearity should be diluted and repeated. The sample may be diluted, repeated and multiplied by the dilution factor automatically by utilizing the AUTO REPEAT RUN.
Interfering Substances:
Results of studies9 show that the following substances interfere with this creatinine procedure.
The criteria for no significant interference is recovery within 10% of the initial value.
Bilirubin: / No significant interference up to 20 mg/dL BilirubinHemolysis: / No significant interference up to 500 mg/dL Hemolysate
Lipemia: / No significant interference up to 700 mg/dL Intralipid*
Protein: / Interference < 20% between 3 and 12 g/dL Protein
* Intralipid, manufactured by KabiVitrium Inc., is a 20% IV fat emulsion used to emulate extremely turbid samples.
In very rare cases gammopathy, especially monoclonal IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
The information presented is based on results from Beckman Coulter studies and is current at the date of publication. Beckman Coulter Inc., makes no representation about the completeness or accuracy of results generated by future studies. For further information on interfering substances, refer to Young10 for a compilation of reported interferences with this test.
Laboratory specific procedure notes:REFERENCES:
1. Tietz, N.W. (ed), Textbook of Clinical Chemistry, W.B. Saunders, 1986.
2. Jaffe, M.Z. Physiol Chem, 10: 391, 1886.
3. Soldin, S.J. et al., Clin Biochem, 11: 82, 1987.
4. Fabiny, D.I. and Ertingshausen, G., Clin Chem, 17: 696, 1971.
5. Cook, J.G.H., Clin Chem Acta, 32: 485, 1971.
6. CLSI/NCCLS. Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline. CLSI Document GP16-A2, 2nd ed. Pennsylvania.
7. Ehret W, Heil W, Schmitt Y, Topfer G, Wisser H, Zawta B, et al. Use of anticoagulants in diagnostic laboratory investigations and stability of blood, plasma, and serum samples. WHO/DIL/LAB/99.1Rev 2.22pp.
8. Tietz, N.W., Fundamentals of Clinical Chemistry, Third Edition W.B. Saunders, 1987.