Creatinine Reagent

INTENDED USE 3. Do not use washed cuvettes. FOR IN VITRO DIAGNOSTIC USE 4. Avoid contact of reagent with eyes, skin and clothing. Do not pipette reagents by Creatinine Reagent is intended for the quantitative determination of creatinine in serum, mouth. Do not ingest. Wash hands after use. plasma and urine. 4. Corrosive (Sodium hydroxide.) Causes burns. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable gloves and SUMMARY eye/face protection. In case of accident or if you feel unwell, seek medical advice Serum creatinine is a waste product formed by the spontaneous dehydration of body immediately creatine. Most of the body creatine is found in muscle tissue where it is present as SPECIMEN COLLECTION creatine phosphate and serves as a high-energy storage reservoir for conversion to Either serum, EDTA, lithium or sodium heparin plasma or urine may be used with the adenosine triphosphate. The rate of creatinine formation is fairly constant with about 2 appropriate system parameter settings. 1 percent of the body creatine being converted to creatinine every 24 hours. Serum Sample collected by standard technique. Creatinine in the serum or plasma sample is creatinine and urea levels are elevated in patients with renal malfunction, especially 8 stable for 24 hours at 2-8°C. Creatinine in the urine sample may be stored at 2-8°C for 4 decreased glomerular filtration. A single, random measurement of serum creatinine may days.8 Both samples may be frozen for longer storage. be used as an indicator of impaired kidney function. In the early stages of kidney damage, the rise in the serum urea levels usually precedes the increase in serum creatinine. This INTERFERING SUBSTANCES advantage is offset by the fact that serum urea levels are affected by factors such as diet, The following compounds at the indicated levels gave less than 0.1 mg/dL interference as degree of hydration, and protein metabolism. Conversely, serum creatinine levels tend to apparent creatinine when added to a normal serum pool. be constant and unaffected by factors affecting serum urea levels. Thus serum creatinine is a significantly more reliable renal function screening test than serum urea.2 A acetoacetic acid 20 mg/dL considerably more sensitive test for measuring glomerular filtration is the creatinine acetone 25 mg/dL 1 clearance test. For this test, a precisely timed urine collection (usually 24 hours) and a ascorbic acid 10 mg/dL blood sample are needed. Urine creatinine levels, as well as other markers of dilution, i.e., albumin 3 g/dL specific gravity and appearance, are useful as an adjunct to drug abuse testing in fructose 200 mg/dL determining external dilution or excessive donor hydration.3,4 Most creatinine assays are glycerol mg/dL based on a modification of the original Jaffe reaction.5 In many of these procedures, the 30 sample is added to an alkaline picrate solution, and the absorbance is read after the glucose 500 mg/dL reaction has reached end point. Although relatively simple and convenient, the end point hemoglobin 10 g/L assays are somewhat non-specific and subject to interferences by serum metabolites. β - hydroxybutyrate 10 mmol/L Improvements in the end point assays have been made by using cation-exchange resins pyruvate 0.2 mmol/L 6 and Lloyd‘s reagent to remove the creatinine from the sample. Though effective, these uric acid 20 mg/dL procedures are time consuming and require centrifugation or filtration steps. More recently, kinetic assays using the Jaffe reaction have been developed to increase the Cephalosporin antibiotic interferences with Jaffe methods for creatinine have been specificity for creatinine and to reduce the interference by serum metabolites.7 described.10 When tested at the concentrations indicated below, the following PRINCIPLE OF PROCEDURE cephalosporin antibiotics spiked into serum containing a level of 1.0 mg/dL creatinine The Creatinine Reagent uses a kinetic modification of the Jaffe reaction. were found to give the amount of interference indicated: Cefaclor at 100 mg/dl showed an increase of 0.7 mg/dL. Alkaline Cefoxitin at 100 mg/dl showed an increase of 5.3 mg/dL. Creatinine + Picric Acid red complex Cephaloridine at 100 mg/dl showed an increase of 1.2 mg/dL. pH Cephalothin at 100 mg/dl showed an increase of 0.2 mg/dL. The rate of formation of the red colored complex is measured at 500 nm on the COBAS Intralipid at 1000 mg/dL (representative of lipemia) caused a negative 0.2 mg/dL ® MIRA and is proportional to the creatinine concentration in the sample. On COBAS interference at a creatinine level of 1.5 mg/dL. Urea at 1000 mg/dL caused a positive 0.2 instruments, a time interval for measuring the reaction is chosen to minimize non- mg/dL interference at a creatinine level of 1. 2 mg/dL. creatinine interferants. Bilirubin interference as apparent creatinine was tested using both conjugated and REAGENTS unconjugated bilirubin spiked samples. For every 1.0 mg/dL of unconjugated bilirubin Reactive ingredients present in the sample, an apparent loss of 0.03 mg/dL creatinine was observed. For every Reagent 1 1.0 mg/dL of conjugated bilirubin (ditaurobilirubin) present in the sample, an apparent loss Sodium Borate 215 mmol/L of 0.08 mg/dL creatinine was observed. Thus, care should be taken in the interpretation of pH 13.1 low creatinine values when high levels of bilirubin are present.

Less than 10% interference was demonstrated from up to 600 mg/dL Hemoglobin F. Reagent 2 Young gives a list of drugs and other substances that interfere with the determination of Picric Acid 52 mmol/L creatinine.11,12 Sodium Borate 35 mmol/L pH 8.0 PROCEDURE Nonreactive ingredient Materials provided Stabilizers R85559 R85559V1 R1 2 x 100 mL The final reaction mixture contains R85559V2 R2 2 x 100 mL Sodium Borate 96 mmol/L Picric Acid 20 mmol/L R85546 R85546V1 R1 10 x 3 mL pH 13.0 R85546V2 R2 10 x 3 mL Nonreactive ingredients Materials required but not provided Stabilizers 1. COBAS MIRA® chemistry analyzer. REAGENT PREPARATION 2. Multi-Analyte Serum Calibrator (R60010) Mix equal volumes of the Reagent 1 and Reagent 2 prior to use. TEST PROCEDURE 30 oC Pour sufficient working reagent into the proper position on the reagent rack and place on 15 oC STORAGE AND STABILITY the instrument. R85546 contains vials that are COBAS MIRA® system rack compatible. Store the reagents at 15-30 °C. The reagents are stable until the expiration date on the See appropriate operator’s manual for further details on system programming and label. Keep reagent bottles tightly closed when not in use. Once opened, Reagent 1, operation. Reagent 2, and combined (working) Creatinine Reagent are stable for 21 days when stored in a tightly closed container and 6 hours when exposed to atmosphere. Discard CALIBRATION the reagent if it appears turbid or fails to recover stated values for the control sera. Use Multi-Analyte Serum Calibrator (R60010) for serum samples. Consult the package Reagent 1 should be clear and colorless; Reagent 2 and the working reagent should be insert for this product for instructions on use. Calibrate in accordance with the instrument clear and yellow in color. manufacturer’s specifications. Calibration stability is dependent upon the instrument performance and the proper storage of the reagents. Re-calibration is recommended at WARNINGS AND PRECAUTIONS anytime, should one of the following occur. 1. Good laboratory safety practices should be followed when handling any 1. Change in the reagent lot number. laboratory reagent. Refer to a recognized laboratory safety program for 2. Preventative maintenance is performed on the analyzer. additional information. (See GP17-T, Clinical Laboratory Safety; Tentative 3. A critical element of the analyzer is replaced. Guideline (1994), National Committee on Clinical Laboratory Standards, 4. Control material results have shifted or are out of range and the use of a freshly Wayne, PA.) reconstituted vial of control does not correct the situation. 2. Results should be interpreted considering all other test results and clinical Each laboratory should establish its own procedures for corrective action if status of the patient. calibration is not acceptable.

QUALITY CONTROL The following table lists the data obtained in a comparison of urine samples with the Controls are recommended to monitor the performance of manual and automated assay reagent for Creatinine (y) with a similar creatinine reagent (x) run on a COBAS MIRA®. procedures, providing a continued screening of the instrument, reagents and techniques. Number of sample pairs (n) 56 Assayed Control Serum Level 1 (R83082) and Assayed Control Serum Level 2 (R83083) Slope 0.94 are recommended for this purpose. Each laboratory should establish its own control y-Intercept -0.694 schedule. Correlation coefficient 0.998 CALCULATIONS Precision COBAS chemistry instruments calculate a factor using the following formula: Precision studies using control sera were performed on a COBAS MIRA according to the 1 CSTD11 CSTD21 13 F= - + guidelines of NCCLS Publication EP5-T. 2 ∆A - ∆A ∆A - ∆A STD11 RB STD21 RB Total Within Run Mean (mg/dL) CV% CV% then: Level 1 4.0 2.7 1.6 Concentration of the sample (mg/dL) = (∆ASample - ∆ARB) x F Level 2 9.4 2.0 0.5 where: C = Concentration in mg/dL Urine specimens with low and high concentrations of creatinine on a COBAS MIRA STD11 = Standard One (first replicate) showed the following performance: STD21 = Standard One (second replicate) Total Within Run ∆A = Absorbance Change Mean (mg/dL) n CV% CV% RB = Reagent Blank Level 1 72.7 20 3.6 3.6 F = Factor Level 2 136.3 20 4.5 2.5 CREATININE CLEARANCE The creatinine clearance is calculated using the following formula: EXPECTED VALUE U 1.73 A study was conducted on a total of 100 samples from normal blood donors (52 females Creatinine clearance (mL/min) = x V x and 48 males) using the Creatinine Reagent. The expected values were found to be 0.7 - 1.4 mg/dL for the female donors and 0.9 - 1.5 mg/dL for the male donors. In a creatinine P A clearance study consisting of 18 donors, the expected values were found to be 69 -149 where: ml/min. U = Concentration of creatinine in the urine P = Concentration of creatinine in the serum or plasma A study was conducted on a total of 50 single, random urine samples from normal donors V = Volume of urine in ml/min using the Creatinine Reagent. The range of values was found to be 15.8 - 260.8 mg/dL A = Body surface area in square meters with a mean of 130.8 mg/dL and a SD of 64.8 mg/dL. Five values were below 30 mg/dL 1.73 = Average body surface in square meters and one was below 20 mg/dL.

It is recommended that each laboratory establish their normal range. The patient’s body surface may be calculated using the following formula or obtained 1 more conveniently from an available nomogram. REFERENCES log A = (0.425 log W) + (0.725 log H) – 2.144 1. Tietz NW. Fundamentals of Clinical Chemistry. Philadelphia, PA: WB Saunders where: Company 1987; 673-684,942. A = The body surface area in square meters 2. Henry JB. Clinical Diagnosis and Management by Laboratory Methods, 16th ed. WB W = The weight of the patient in kg Saunders, Philadelphia, PA, 1974; 263. H = The height of the patient in cm 3. Needleman SB, Porvaznik M, Ander D. Journal of Forensic Sciences. 1992; 37 (4):1125. PROCEDURAL LIMITATIONS 4. Mayer B, Hemphill G. Clin Chem. 1992; 38 (6) 1008 Samples with creatinine values greater than 30 mg/dL for the serum application or 200 5. Jaffe M. Z Physiol Chem. 1886; 10: 391. mg/dL for the urine application should be diluted with 0.9% NaCl or deionized water and re-assayed. Correct the results for the dilution factor. 6. Henry RJ, Cannon DC, and Winkelman JW. Clinical Chemistry; Principles and Techniques, 2nd ed. Harper and Row, Hagerstown, MD, 1974;541 Cobas MIRA® systems with automatic post-dilution extend the serum and plasma assay 7. Larsen K. Clin Chem Acta. 1972; 41:209. linearity to 60 mg/dL. 8. Tietz NW. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia PA: WB A result obtained with this reagent should serve only as a diagnostic aid and is not to be Saunders Company 1995:186-188. interpreted as diagnostic in and of itself. 9. Data on file at Cliniqa Corporation. PERFORMANCE CHARACTERISTICS 10. Swain R, Briggs S. Clin Chem. 1977;23:1340-1342. Linearity 11. Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests, 2nd ed. Washington DC: AACC Press 1997; 3.161 – 3.171. The assay is linear to 30.0 mg/dL for the serum and plasma application and 200 mg/dL 12. Young DS. Effects of Drugs on Clinical Laboratory Tests. 4th ed. Washington, DC: using the urine application. AACC Press, 1995. Accuracy 13. National Committee for Clinical Laboratory Standards. User Evaluation of Precision Comparison of Creatinine Reagent (y) runs on the COBAS MIRA system and another Performance of Clinical Chemistry Devices; Tentative Guideline. Villanova, PA: commercially available picric acid method. NCCLS; 1984:4(8). NCCLS Publication EP5-T.

Number of sample pairs (n) 105 Slope 1.06 y-Intercept 0.14 Correlation coefficient 0.99

For in vitro diagnostic use See package insert for proper use

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