ENZYMATIC CREATININE Insert

Transcription

1 ENZYMATIC CREATININE Insert Ref.:17 Intended use. System for determination of creatinine in serum, plasma, and urine samples by end-point reaction. Professional use. [ Only for in vitro diagnostic use.] Test principle. Creatinine present in sample is converted into creatine by creatinine amidohydrolase. The creatine produced is hydrolyzed to sarcosine and urea by creatine amidinohydrolase. Next, the enzyme sarcosine oxidase causes the oxidative demethylation of sarcosine, yielding glycine, formaldehyde and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide reacts with N-ethyl-Nsulfopropryl-m-toluidine (ESPMT) and 4-aminoantipyrine, yielding a quinoneimine with maximum absorbance at 546 nm. The color intensity of the reaction product is directly proportional to the creatinine concentration in sample. Creatinine + H O Creatine + H O Creatinine amidohydrolase Creatine amidinohydrolase Creatine Sarcosine + Urea Sarcosine oxidase Sarcosine + H O + O Glycine + Formaldehyde + H O Peroxidase H O + ESPMT + 4-aminoantipyrine Quinoneimine + 4H O Summary. Enzymatic Creatinine Labtest uses the enzymes creatinine aminohydrolase, creatine amidinohydrolase, and sarcosine oxidase coupled with the Trinder reaction to determine the creatinine concentration in serum, plasma, and urine samples. The enzymatic methodology makes the analyte determination more specific, eliminating interference from plasma proteins and other chromogens commonly observed in direct method based on the Jaffé reaction. The calibration material indicated is calibrated with NIST SRM 914, which makes the method traceable to the IDMS (isotopic dilution mass spectrometry) definitive method. Therefore, the method meets the recommendations from the National Kidney Disease Education Program (NKDEP) for standardization of creatinine determination in serum. Methodology. Enzymatic Trinder. Reagents Reagent 1- Store at - 8 º C. Contains buffer ph 7.4; creatine amidinohydrolase IU/mL; sarcosine oxidase 17 IU/mL; ascorbate oxidase <7 IU/mL; and N-ethyl-Nsulfopropryl-m-toluidine 0.1 mg/ml.. - Reagent - Store at - 8 º C. Contains buffer ph 7.3; creatinine amidohydrolase 670 IU/mL; peroxidase 91 IU/mL; 4-aminoantipirine 0.9 mg/ml and sodium azide <0.1% The reagents must be kept out of their storage temperature for only the time necessary to obtain the volume to be used in tests. Avoid direct sun light exposure. The unopened reagents, when stored at indicated temperature, are stable up to the expiration date shown on the label. Upon handling, reagents and the calibrator may be submitted do microbial or chemical contamination, which may cause a reduction in reagent stability. Precautions and warnings The usual security cares should be applied to the reagent handling. They must not be pipetted by mouth aspiration. Avoid ingestion and in case of contact with eyes, wash them with plenty of water and seek medical help. Reagent contains sodium azide as preservative. Avoid ingestion. In case of contact with eyes, immediately flush eyes with plenty of water and get medical assistance. Sodium azide may react with lead and copper plumbing and yield highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide accumulation. Materials required not provided 1. Analyzer capable of measuring absorbance accurately at 546 nm ( nm).. Calibrator from the Calibra H Labtest series. 3. Pipets to measure samples and reagents. 01 English - Ref.: 17

2 Specimen collection and preparation A Standard Operating Procedure (SOP) must be created to establish adequate procedures for sample collection, preparation, and storage. The errors due to bad sampling can be more damaging than the ones which may occur during the analytical procedure. Use serum or plasma (heparin, EDTA, fluoride, oxalate, and citrate) samples. The analyte is stable for 7 days at - 8 º C. The Glistab Labtest (Ref. 9) allows for collection of only one sample for determination of creatinine, glucose and urea. 4-hour urine samples must be centrifuged. The urine sample must not receive any preservative, and must be stored under refrigeration during the collection period and after it is received by the laboratory. Since no known test method can offer complete assurance that human samples will not transmit infectious diseases, all samples should be considered potentially infectious and handled accordingly. Disposal of all biological waste material should be in accordance with local guidelines. Interference Concentrations of triglycerides up to 1000 mg/dl, bilirubin (conjugated and unconjugated) up to 16 mg/dl, hemoglobin up to 400 mg/dl, ascorbic acid up to 40 mg/dl, creatine up to 0 mg/dl, cefpiramide up to 100 mg/dl, cefotaxime up to 100 mg/dl, and ceftraixone up to 100 mg/dl do not interfere in sample testing. Dobutamine and methyldopa interfere negatively in the reaction. Samples with bilirubin, hemoglobin, and triglycerides levels higher than the ones indicated above must be diluted with NaCl 150 mmol/l (0.85%) prior to being tested. Samples with azide may yield inaccurate results for creatinine caused by insufficient creatine conversion. To determine the approximate concentration of hemoglobin in a sample, dilute 0.05 ml of sample in.0 ml of NaCl 150 mmol/l (0.85%) and measure the absorbance at either 405 or 415 nm, subtracting the zero absorbance value with deionized water. Procedure To determine the creatinine concentration in urine, dilute the sample 1:5 (0. ml of urine ml of NaCl 150 mmol/l). Multiply the result obtained by 5. Parameter Bi-Reagent Application Reaction Type End-point Reaction Direction Increasing Primary Wavelength 546 nm Secondary Wavelength 800 nm Temperature 37 ºC Points Calibration Point 0: NaCl 0.85% or Deionized water Point 1: Calibra H Calibration Model Linear Sample Volume* 6 L R1 Volume* 70 L Reading 1 (Abs 1) 300 seconds after incubating R1 at 37 º C + sample R Volume* 90 L Reading (Abs ) 300 seconds after incubating R1 at 37 º C + sample + R * Sample and reagent volumes can be modified proportionally without any loss in test performance, and the calculation procedure remains the same. In case of volume reduction it is crucial to observe the minimal necessary volume for photometric reading. Calibration Automated systems -point Calibration Point 0: Reagent blank - deionized water or NaCl 150 mmol/l (0.85%). Point 1: Calibrator - Calibra H Labtest series. The creatinine concentration in the Calibra H material is traceable to the Standard Reference Material (SRM) 914 from the National Institute of Standards and Technology (NIST). Calibration frequency When the internal quality control indicates so; When using a new reagent lot; When using new bottle of reagent from the same lot if a new calibration has been performed for the prior reagent bottle. Calculation. According to recommendations of NKDEP the results must be reported with two decimal places to avoid systematic errors caused by rounding of results, which may reach 6%. Abs of Test or Calibrator = Abs - Abs 1 Abs Test Creatinine (mg/dl) = x Calibrator conc. mg/dl Abs Calibrator Urine Creatinine Urine Creatinine (mg/dl) Urine Creatinine = x Urine volume (mg/4 h) 100 (ml/4 h) mg/kg weight = mg/4 hours divided by body weight. 0 English - Ref.: 17

3 Endogenous creatinine clearance. Inform the patient so they can collect a 4-hour urine sample. Determine the creatinine concentration in serum and urine. The serum sample can be obtained in any moment during the period of urine collection. Apply the results found to the equation below: Clearance = U S x MV (ml/minute) U: urine creatinine (mg/dl) S: serum creatinine (mg/dl) MV: minute volume (4-hour urine volume in ml divided by 1440). Note: The clearance results must be corrected according to the patient's body surface area, which is obtained via a nomogram that correlates weight and height, or using the equation below: A = W x H x A = body surface area (m ) W = weight (kg) H = height (cm) Multiply the clearance value by 1.73 and divide the result by the patient's body surface area. Glomerular filtration rate. The NKDEP strongly recommends that laboratories report the estimated glomerular filtration rate (egfr) for all creatinine results. When the results for plasma creatinine are traceable to the IDMS method, the following equations are applied, which use creatinine (CREA), age (18 to 70 years) and sex. Women egfr ( ml/min/1.73m) = 175 * (CREA) * (Age) * Men egfr ( ml/min/1.73m) = 175 * (CREA) * (Age) According to recommendations from the NKDEP, egfr must be reported as calculated value when the result is equal o less than 60 ml/min/1.73m. When the calculated value is higher than 60, it must be reported as either higher than 60 ml/min/1.73m or >60 ml/min/1.73m. It is recommended to use the products Qualitrol H - Labtest as internal quality control. It is recommended to meet the specification proposed by the NKDEP for coefficient of variation 4% and systematic error 5%. Expected values. These values should be used only for orientation purposes. Each laboratory should evaluate the transferability of the expected values to its own patient population and, if necessary, estimate its own reference interval. Serum/Plasma (mg/dl)* Newborn weeks - 1 year <3 years <5 years <7 years <9 years <11 years <13 years <15 years Adults (women) years Adults (men) years * Intervals established for results traceable to the IDMS method. There are no intervals established for patients between 15 and 18 years old. It is suggested to use the intervals established for adult men and women. Conversion of mg/dl to SI units: mol/l = mg/dl x 88.4 Urine (mg/kg/4 hours) - 3 years 6 - > 3 years 1-30 Adults (women) 16 - Adults (men) 1-6 Creatinine Clearance (ml/min/1.73m )** Children Adults (women) Adults (men) **Intervals established for results traceable to the IDMS method. Operating interval. The reaction is linear between 0.0 mg/dl and 150 mg/dl. For higher concentrations, dilute the sample with NaCl 150 mmol/l (0.85%), perform a new test, and multiply the result obtained by the dilution factor used. The NKDEP recommends the calculation of glomerular filtration rate (egfr) instead of creatinine clearance, using the creatinine result traceable to the IDMS method. Internal quality control. The laboratory must keep an internal quality control program with well-defined regulations, objectives, procedures, criteria of quality specifications and tolerance limits, corrective actions and registration of activities. materials should be used for measurement imprecision monitoring and determination of calibration deviation. 03 English - Ref.: 17

4 Performance characteristics Recovery studies. Two samples with creatinine concentrations equal to 0.3 mg/dl and mg/dl received 0.3 mg/dl of creatinine, yielding recovery rates between 99.5% and 100.3%. The proportional systematic error, estimated based upon the decision level equal to 1.0 mg/dl, is mg/dl or 0.1%. Method comparison. The Enzymatic Creatinine method was compared against a similar method, and the following results were obtained: For serum samples: Comparative Method Enzymatic Creatinine Sample Nature Serum Sample Number Concentration interval (mg/dl) Estimate mean (mg/dl) Regression equation Enzymatic Creatinine = x Comparative Correlation coefficient Decision levels for creatinine evaluation Imprecision Creatinine estimated Systematic errors using the regression estimated based on equation creatinine decision levels mg/dl mg/dl mg/dl % Imprecision - Within Run N Mean SD CV (%) Sample Sample Imprecision - Run-to-Run N Mean SD CV (%) Sample Sample Sample Using the regression equation, the following systematic errors (bias) were found for the Enzymatic Creatinine method: Decision levels for creatinine evaluation For urine samples: Creatinine estimated Systematic errors using the regression estimated based on equation creatinine decision levels mg/dl mg/dl mg/dl % Comparative Method Urine Enzymatic Creatinine Sample Nature Sample Number Concentration interval (mg/dl) Estimate mean (mg/dl) Regression equation Enzymatic Creatinine = x Comparative Correlation coefficient Using the regression equation, the following systematic errors (bias) were found for the Enzymatic Creatinine method: Methodology sensitivity. A sample containing no creatinine was used to evaluate the assay's detection limit. The value found was 0.19 mg/dl, which corresponds to the mean value of 9 assays plus two standard deviations. Using the standard's absorbance as parameter, the photometric detection limit was equal to 0.04 mg/dl, which corresponds to a difference in absorbance equal to Effect of matrix dilution. A sample with concentration of mg/dl was used to evaluate the system response to matrix dilution using NaCl 150 mmol/l. Using dilution factors ranging from 1.5 to 5, the recovery values found were between 99.5% and 100.7% Notes 1. The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results.. The clinical laboratory is aimed at providing accurate and precise results. Use of inappropriate quality water is a potential cause of analytical bias. The water used in the laboratory should have the appropriate quality for each application. Thus, to prepare reagents, use in the measures and for use in the final rinsing of the flasks, the water should have resistivity 1 megaohm.cm or conductivity 1 microsiemens/cm and silicate concentration <0,1 mg/l. When the deionizing column is with its capacity saturated, occurs release of several ions, silicates and substances with large oxidation or reduction power that deteriorate the reagents in a few days or even hours, thus changing the results unpredictably. Consequently, it is essential to establish a quality control program for the water. 04 English - Ref.: 17

5 References 1. JUNGE, W. et. al. Determination of reference intervals for serum creatinine, creatinine excretion and creatinine clearance with an enzymatic and a modified Jaffé method. Clinica Chimica Acta, v. 344, n. 1-, p , MYERS. G. L. et al. Recommendations for Improving Serum Creatinine Measurement: A Report from the Laboratory Working Group of the National Kidney Disease Education Program. Clinical Chemistry, v. 5, n. 1, p. 5-18, MARTENSSON, A. et. al. Creatininium reference intervals for corrected methods. Scandinavian Journal of Clinical and Laboratory Investigation, v. 64, p , CERIOTTI, F. et. al. Reference Intervals for serum Creatinine Concentrations: Assessment of Available Data for Global Application. Clinical Chemistry, v.54, n. 3, p , Labtest: Data on file. Application procedures using Enzymatic Creatinine are available for various automated instruments. Customer information [Warranty conditions] Labtest Diagnóstica warrants the performance of this product under the specifications until the expiration date shown in the label provided that the procedures and storage conditions indicated on the label and in this insert have been followed correctly. Labtest Diagnóstica S.A. CNPJ: / Av. Paulo Ferreira da Costa, Vista Alegre - CEP Lagoa Santa. Minas Gerais Brasil - Customer Service customerservice@labtest.com.br Product Reference Content 1 X 16 ml 17-1/16 Enzymatic 1 X 54 ml Creatinine X 54 ml 17-/7 X 18 ml Enzymatic Creatinine X 54 ml 17-/7 Labmax 560/400 X 18 ml Presentation Edition: October, 01 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 05 English - Ref.: 17

6 Símbolos utilizados com produtos diagnósticos in vitro Símbolos usados con productos diagnósticos in vitro Symbols used with ivd devices Conteúdo suficiente para < n > testes Contenido suficiente para < n > tests Contains sufficient for < n > tests Risco biológico Riesgo biológico Biological risk Data limite de utilização (aaaa-mm-dd ou mm/aaaa) Estable hasta (aaaa-mm-dd o mm/aaaa) Use by (yyyy-mm-dd or mm/yyyy) Marca CE Marcado CE CE Mark Calibrator Material Tóxico Tóxico Poison Calibrator Material Reagente Reactivo Reagent Limite de temperatura (conservar a) Temperatura limite (conservar a) Temperature limitation (store at) Fabricado por Elaborado por Manufactured by Representante Autorizado na Comunidade Europeia Representante autorizado en la Comunidad Europea Authorized Representative in the European Community Número do lote Denominación de lote Batch code Consultar instruções de uso Consultar instrucciones de uso Consult instructions for use e Número do catálogo Número de catálogo Catalog Number e negativo negativo Negative control Adições ou alterações significativas Cambios o suplementos significativos Significant additions or changes e positivo positivo Positive control Produto diagnóstico in vitro Dispositivo de diagnóstico in vitro In vitro diagnostic device e Liofilizado Liofilizado Lyophilized Corrosivo Corrosivo Corrosive Período após abertura Período post-abertura Period after-opening Uso veterinário Uso veterinario Veterinary use Instalar até Instalar hasta Install before Ref.: English - Ref.: 17