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Intended Use . Test Principle . Summary . Methodology . Reagents 1 Intended use . Colorimetric system for creatinine determination in 3. - Standard 4.0 mg/dL - Store at 15 - 25 ºC. blood and urine by end point reaction. Reagent label bears expiration date. In order to avoid evaporation of the Standard, keep the bottle tightly closed. Test principle . Creatinine and other components of serum react to alkaline picrate in alkaline solution yielding a red complex what is 4. - Acid solution - Store at 15 - 25 ºC. photometrically measured. Acetic acid (11.4 mol/L). The addition of an acid decrease the pH to 5.0 yielding the decomposition of the creatinine picrate, the chromogen derived color remained unaltered Precautionsandwarnings what is also a photometric measurement. The difference of the two measurements yields the true value of creatinine. For in vitro diagnostic use. Disposal of all waste material should be in accordance with local Summary . Creatinine applies an end point procedure in order to guidelines. improve the method specificity and minimize the susceptibility to 1-3, 5 interferences . The usual security cares should be applied on the reagent handling. The measurement procedure is calibrated with the NIST SRM 914 and The reagents Buffer and Acid solution are corrosive. Avoid ingestion. In renders the results traceable to the IDMS (isotropic dilution, mass case of eyes, skin and mucosa contact, immediately flush then with plenty spectrometry) definitive method, which complies the National Kidney of water and get medical assistance. Disease Education Program (NKDEP) recommendations for standardization of serum creatinine measurement4 . The Buffer may result in ulcerations when ingested. In case of ingestion, immediately offer a lot of water with lemon juice or vinegar. Do not induce All the direct methods that apply the Jaffe reaction are susceptible to a vomiting and get medical assistance. constant systematic error, due to the plasmatic proteins ant other chromogens interference. In order to minimize this error and increase the In case of ingestion of Picric Acid, offer 4 glasses of water and if the Creatinine results accuracy, Labtest recommends the use of the individual is conscious, induce vomiting and get medical assistance. correction index that must be applied whatever are the found results8,11 . Storage and stability . Unopened reagents, when stored at Several studies have demonstrated that is possible minimize significantly indicated temperature, are stable up to expiration date shown on the label. the interferences caused by icteric and lipemic samples in creatinine measurement3 . The procedure with acidification step minimizes the Deterioration . Microbial or chemical contamination may decrease bilirubin negative interference, while the desproteinization procedure reagents stability. removes interference of lipemic samples equivalent to a triglycerides 11 value of 250 up to 1800 mg/dL . Specimencollectionandpreparation The measurement procedure is applied in automated and semi- Use serum or plasma (heparin, EDTA, fluoret, oxalate and citrate). The automated systems able to perform accurate measure of absorbance at analyte is reportedly stable for about 7 days at 2 - 8 ºC. 510 nm. Glistab anticoagulant (Labtest Ref.: 29) allows the collection of only one Methodology . Labtest. blood sample for the measurements of creatinine, glucose, and urea. Reagents Urine of 24 hours and amniotic fluid must be stored at 2 - 8 ºC during the period of collection until it is measured. 1. - Picric acid - Store at 15 - 25 ºC. No known test method can offer complete assurance that human blood Reagent label bears expiration date. Picric acid 44.4 mmol/L. samples will not transmit infectious diseases. Therefore, all blood derivatives should be considered potentially infectious. 2. - Buffer - Store at 15 - 25 ºC. Reagent label bears expiration date. Sodium hydroxide (208 mmol/L), sodium tetraborate (12.7 mmol/L) and surfactant. The reagent may Interference precipitate in low temperatures at 15 ºC. In this case, warm up at 37 ºC Proteins present in the samples yield a positive interference introducing a and mix until it dissolves completely. constant systematic error. This error may be minimized applying a correction index. 01 English-Ref.:35 Since the urine has no proteins that may interfere, the correction index is Calculations not applied to the calculation of concentration in urine samples. See application of correction index on Calculations. A-A1 2 Creatinine (not corrected) = x 4 mg/dL Creatinine determination in urine samples may be affected by the action of Astandard high amount of reducers substances present in cases of ketoacidosis. Boiling the urine sample for one minute eliminates partially these According with NKDEP4 recommendations, the results should be reported substances interference. The remaining interference is excluded in the with two places of decimals in order to avoid systematic errors due to procedure with acidification step. making the results round, which may reach ± 6%. For therapeutic control it is recommended to collect the sample at the Due the great reproductive results of the assays system, it is possible to same time due circadian variations. use the factor method: The aspirin in anti-inflammatory doses increases the creatinine value in blood sample. Calibration factor = 4 /Astandard Physical exercises increase the creatinine values. Creatinine values are lower in individuals who have vegetarian diet. Creatinine (not corrected) = (A1 - A 2 ) x Factor (mg/dL) Bilirubin up to 5 mg/dL, hemoglobin up to 180 mg/dL and triglycerides up Urinary creatinine to 250 mg/dL do not interfere significantly. Bilirubin values over 5 mg/dL interfere negatively in the reaction. Urinary Creatinine Urinary (mg/24 hours) = x UrineVolume (mL/24h) Triglycerides values over 250 mg/dL provide false increased results. Creatinine 100 Materials required not provided mg/kg weight = mg/24 hours divided by body weight 1. A constant temperature water bath (37 ºC). Applying the correction index . Plasmatic proteins interference 2. Photometer capable of measuring absorbance at 500 - 540 nm. 5 that occurs in Jaffe reaction, introduce a constant error in the 3. Pipettes to measure reagents and samples. measurement which is minimized by the correction index (0.25 mg/dL). 4. Timer. The obtained results with the calibration and the correction are traceable to IDMS method and comply with the NKDEP4 Manualanddirectassayprocedure recommendations. See notes 1, 2 and 3. Creatinine=Creatinine-Correctionindex (corrected) (not corrected) (0.25 mg/dL) Urine . Dilute the urine 1:25 with distilled water (0.2 mL of urine + 4.8 mL of distilled water) and multiply the result by the dilution factor (25). Procedurewithdeproteinization The water must have resistivity≥ 1 megaohm, or conductivity ≤1 microsiems and silicates concentration must be <0.1mg/L. This must be applied to icteric and turbid samples. Set up three tubes and proceed as follows: Mix 0.5 mL of serum or plasma to 1.0 mL of Picric Acid (n° 1), homogenize and centrifuge 10 minutes. Blank Unknown Standard Set up three tubes and proceed as follow Buffer(Nº.2) 2.0mL 2.0mL 2.0mL Sample 0.25mL Distilledordeionizedwater 0.25mL Blank Unknown Standard Standard(Nº.3) 0.25mL Buffer(Nº.2) 2.0mL 2.0mL 2.0mL Picricacid(Nº.1) 0.5mL 0.5mL 0.5mL Supernatant 0.75mL Distilledordeionizedwater 0.25mL Standard(Nº.3) 0.25mL Mix and incubate in a water bath at 37 ºC during 10 minutes. Measure the Picricacid(Nº.1) 0.5mL 0.5mL absorbance of the Unknown and Standard against Blank at 510 nm or green filter (500 - 540 nm). Mix and incubate in a water bath at 37 ºC during 10 minutes. Measure the Absorbance will be A1 . absorbance of the Unknown and Standard against Blank at 510 nm or green filter (500 - 540 nm). Acidsolution(Nº.4) 0.1mL 0.1mL Absorbance will be A1 . Mix and let at room temperature for 5 minutes. Measure the absorbance of the Unknown against Blank at 510 nm or green filter (500 - 540 nm). Acidsolution(Nº.4) 0.1mL 0.1mL Absorbance will be A2 . 02 English-Ref.:35 Mix and let at room temperature for 5 minutes. Measure the absorbance of Manual calibrations the Unknown against Blank at 510 nm or green filter (500 - 540 nm). Perform a new calibration after reagent lot change or when the internal quality control indicates. Absorbance will be A2 . Use the same calculation proposed for the direct assay. Do not apply the Quality control . For quality control use Qualitrol H Level 1 and correction index. Qualitrol H Level 2 or other suitable control material. The limits and control In turbid samples in excess, it is not possible to get a clear supernatant in interval must be adapted to the laboratory requirements. the deproteinization procedure. In this case, it is not possible to measure Each laboratory should establish corrective actions to be taken if values the creatinine. fall outside the control limits. Endogenous creatinine depuration. The laboratory must Measurement/reportablerange inform the patient how to collect the correct urine within 24 hours. The reaction is linear between 0.2 mg/dL and 12 mg/dL. Use the proposed methodologies to measure creatinine in serum and urine. If creatinine concentration exceeds 12 mg/dL, the sample must be diluted with 0.85% NaCl. Multiply the result by the appropriate dilution factor. Apply the obtained results in the following equation: Expected values . Each laboratory should evaluate the U transferability of the expected range to its own patient population and, if Depuration (mL/minute) = x VM 8,10 S necessary, estimate its own
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