GLUCOSE GOD. 01 English - Ref.: 134. Ref.:134. Insert

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1 GLUCOSE GOD Insert Ref.:134 Intended use. Enzymatic system for the determination of glucose in blood, cerebrospinal, ascitic, pleural and synovial fluids by kinetic method. [ Use only for in vitro diagnosis. ] Test principle. Glucose oxidase catalyses the oxidation of glucose according to the following reaction: Glucose + O + H O GOD Gluconic acid + H O Hydrogen peroxide formed reacts with 4-aminoantipyrine and phenol, under the catalyst action of peroxidase by means of an oxidative coupling reaction forming the red color antipyrilquinoneimine whose intensity is proportional to the concentration of glucose in the sample. 2H O + 4-aminoantipyrine + phenol POD Antipyrilquinoneimine + 4H O Summary. Reagent is ready for use and employs enzymatic methods of high analytical specificity, simple and easy to use in the clinical laboratory. Labtest developed the Glucose GOD system optimizing the reagent enzyme concentrations in order to provide the best analytical performance and higher stability. Repeatability and reproducibility data obtained with the Glucose GOD system show that the method is capable of delivering results that go beyond the performance targets for glucose measures established by the American Diabetes Association (ADA). A comparison between the inaccuracies found in repeatability and reproducibility shows that the measuring system is very robust in regions of significant concentrations for clinical use, indicating it has a very stable performance in routine use. Reagent was developed for use in automated systems. The glucose determination is performed by a kinetic method, obtaining results in a short period of time, which provides more agility for certain biochemical analyzers. The method is easily applicable to automated analyzers capable to accurately measure the absorbance between 490 and 520 nm. Methodology. GOD - Trinder. Reagents Reagent 1 - Store at 2-8 ºC. Contains phosphate buffer at 30 mmol/l, ph 7.5, phenol at 1 mmol/l, glucose oxidase 1000 U/L, peroxidase 80 U/L, 4-aminoantipyrine at 290 µ mol/l, sodium azide at 7.5 mmol/l, and surfactants. Unopened reagent, when stored under the recommended conditions, is stable up to the expiration date printed on the label. During handling, the reagents are subject to contamination of chemical and microbial nature, which may cause reduction in stability. Precautions and warnings Usual safety care should be applied in the reagent handling. The reagent contains sodium azide which is toxic. Caution should be taken to avoid ingestion, and in case of eye contact, wash immediately with plenty of water and seek medical attention. Azide can form highly explosive compounds with lead and copper pipes. Therefore, use large volumes of water to discard the reagent. Do not use Reagent 1 if its measured absorbance against water at 505 nm is equal to or greater than or when it appears cloudy or with contamination signs. Materials needed and not provided 1. Water bath maintained at constant temperature (37 º C). 2. Analyzer capable of measuring absorbance between 490 and 520 nm. 3. Pipettes to measure samples and reagent. 4. Stopwatch. Pre-analytical influences. As it is a substance with reductive features, ascorbic acid prevents the formation of chromogen leading to obtaining falsely decreased results. Patients who take ascorbic acid (Cebion, Energil C, Redoxon, among others), should be advised to 9 discontinue the use for 24 hours before the exam. A significant reduction in blood glucose occurs in the 24 hours following the acute ingestion of alcohol. The reductions may also be significant in individuals undergoing prolonged fasting or in obese individuals treated 3 with low-calorie diets. Diabetic patients on continued use of chlorpropamide (Diabinese) may develop significant hypoglycemia, which is very difficult to adjust. The intra-individual biological variation of glucose is 5.7% and the intragroup biological variation is 6.9% English - Ref.: 134

2 Specimen collection and preparation It should be established a Standard Operating Procedure (SOP) for collection, preparation and storage of the sample. We emphasize that errors derived from sample can be much greater than the errors occurred during the analytical procedure. Blood samples should be obtained after fasting for at least 8 hours or a shorter period according to medical recommendation. Use plasma or serum by taking the following precautions: Perform blood collection by using anticoagulant containing a gycolysis inhibitor. The use of the anticoagulant Glistab (Labtest Ref. 29) allows the collection of one sample for measurements of creatinine, glucose and urea. Blood samples not containing antiglycolytic agent should be centrifuged immediately after collection, and the plasma or serum separated from cells or clot. In other biological fluids (cerebrospinal fluid and ascitic, synovial and pleural fluids), add anticoagulant containing antiglycolytic agent at the same ratio used for the blood sample and centrifuge before starting the 6 measurement. In blood samples treated with antiglycolytic agent, glucose concentration remains stable up to 8 hours. In plasma, serum and other fluids separated from cells, the glucose remains stable for 3 days at 2-8 º C, when there is 6 no bacterial and fungal contamination. Since no known test can ensure that blood samples do not transmit infection, all samples must be considered potentially infectious. Therefore, when handling them, follow the established standards for biosafety. To discard the reagents and biological materials, we suggest applying the local, state or federal environmental protection standards. Interference Hemoglobin concentrations up to 200 mg/dl and triglycerides up to 1000 mg/dl do not produce significant interferences. Do not use icteric samples for the assay. In order to assess the approximate concentration of hemoglobin in a hemolyzed sample, you may proceed as follows: Dilute 0.05 ml of the sample in 2.0 ml of NaCl at 150 mmol/l (0.85%) and measure the absorbance at 405 or 415 nm, adjusting the zero with deionized or distilled water. Hemoglobin (mg / dl) Absorbance405 x 601 Hemoglobin (mg / dl) Absorbance415 x 467 Procedure Parameters for automated analyzers Parameters Type of Reaction Direction of Reaction Primary λ Secondary λ Temperature Calibration Calibration Model* Sample Volume** Volume of R1** Reading 1 (Absorbance 1) Reading 2 (Absorbance 2) Parameters intended for Labmax 240. R1: 300 µ L Sample: 3.0 µ L 37 ºC nm 30 seconds *The definition of calibration model must be appropriate for each type of equipment. If in doubt, please contact the Labtest Customer Service. **The volumes of sample and reagents can be modified proportionately without prejudice to the test performance. In case of reduced volumes, it is essential to observe the minimum volume required for photometric measurement. Calibration. Application Kinetic Ascending 505 nm 660 nm 37 ºC Two points Point 0: Blank (Deionized water) Point 1: Calibrator 1 Linear 3.0 µ L 300 µ L 30 seconds after addition of sample 300 seconds after the sample A1 300 seconds The analyte concentration in the material Calibra H is traceable to the Standard Reference Material (SRM) 917 of the National Institute of Standards and Technology (NIST). Analytical measurement range. The measurement result is linear up to 600 mg/dl. When obtaining a value equal to or greater than 600 mg/dl, dilute the sample with NaCl at 150 mmol/l, repeat the measurement and multiply the result by the dilution factor. We recommend checking the methodological and photometric linearity at least every six months by using samples with values up to 600 mg/ dl. Q uality control. The laboratory should maintain an internal quality control program, which clearly defines the objectives, procedures, standards, criteria for tolerance limits, corrective actions and activity records. materials should be used to monitor the inaccuracy of measurement and calibration deviations. It is suggested that specifications for the variation coefficient and total error are based on the 4,10 components of biological variation (BV). A2 02 English - Ref.: 134

3 It is suggested to use the products of QUALITROL - Labtest line for internal quality control in clinical chemistry assays. Expected values. Intervals should be used only as a guide. It is recommended that each laboratory establishes its own reference range in the attended population. Plasma (8 hours fasting) Age Premature 0 to 1 day > 1 day Children and adults The criteria for diagnosis of pre-diabetes can be obtained from: American Diabetes Association. Diabetes Care 2006; (suppl 29): S43-S48. CSF: 2/3 of blood glucose when measurement is performed on samples collected simultaneously. In healthy individuals, the small amount of liquid present in joint, pleural and peritoneal cavities originates from plasma ultrafiltrate. Therefore, it can be considered that, practically, the glucose is present at the same concentration as in plasma. Conversion. Conventional units (mg/dl) x = IS Units (mmol/l) Performance characteristics 12 Recovery Studies. Two samples with glucose concentrations equal to 85 and 180 mg/dl were added with different amounts of analyte, obtaining recovery between 102% and 103%. The mean proportional systematic error obtained at 120 mg/dl is equal to 3.0 mg/dl or 2.5%. Method comparison. The method was compared to a similar method having the following results: Number of samples Range of concentrations (mg/dl) Regression equation Correlation coefficient Comparative Method (mg/dl) 20 to to to to 99 By using the regression equation, the total error (constant and proportional) checked at the decision limit (120 mg/dl) was equal to 0.14 mg/dl or 0.12%. The total error obtained in the same decision level is 2.08%. The results for the comparative study meet the specification for Total Systematic Error of 6.9% for Biological Variation. As the samples were randomly selected from outpatients and hospitalized patients, it can be concluded that the method has an adequate methodological specificity and meets the requirements specified by the 5 American Diabetes Association (ADA) Labtest Method Labtest method (mg/dl) = x Comparative Precision studies. Precision studies were carried out using 20 samples with mean concentrations equal to 47, 128 and 196 mg/dl. Imprecision - Within Run Sample 1 Sample 2 Sample 3 Imprecision - Run-to-Run Sample 1 Sample 2 Sample 3 The total error (random error + systematic error) estimated at concentrations equal to 45 mg/dl, 120 mg/dl and 180 mg/dl is equal to 4.76%, 2.08% and 2.23% respectively. The results indicate that the method meets the desired specification for total error ( 6.9%) based on the desired components of the Biological Variation. Analytical sensitivity. A protein sample containing 43 mg/dl of glucose was used to calculate the detection limit for the assay, being found a value equal to 1.83 mg/dl, equivalent to 3-fold the standard deviation of sample replicates. When using the absorbance of the standard as a parameter, the photometric detection limit is 0.28 mg/dl corresponding to an absorbance equal to Effects of matrix dilution. Two samples with values equal to 676 and 669 mg/dl were used to evaluate the system response in dilutions of the matrix with NaCl at 150 mmol/l (0.85%). When using dilution factors ranging from 2 to 8, it was observed recoveries between 102 and 107%. When using the absorbance of the standard as the parameter, the photometric detection limit is 1.12 mg/dl, corresponding to an absorbance of Notes N Mean SD (%) CV N Mean SD (%) CV The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results. 2. The deionized or distilled water in the laboratory to prepare reagents, use in the measurements and for final glass washing must have resistivity 1 megaohm.cm, or conductivity 1 microsiems/cm and silicates concentration must be <0.1 mg/l. 3. Several publications demonstrated that urine contains numerous substances, especially uric acid, which interferes with the methods using GOD-POD reaction, leading to falsely decreased results. 03 English - Ref.: 134

4 References Presentation 1. Bergmeyer HU. Methods of Enzimatic Analysis, 3a. edição, Vol VI, Deerfield Beach:VCH, 1986: Product Equipment Labmax 240 Reference /40 1 Content 10 X 40 ml 2. Blaedel WJ, Uhl JM. Clin Chem 1975;21: Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests, 1a. edição, Washington: AACC Press, Biological Variation Database specifications - Westgard QC. Disponível em:< (acesso em 28/10/2011). Glucose GOD Labmax 560 Linha CS / / / X 65 ml X 70 ml 10 X 70 ml 5. Sachs DB, Bruns DE, Goldstein DE, Maclaren NK, McDonald JM, Parrot M. Clin Chem 2002;48: Tietz NW. Fundamentals of Clinical Chemistry, Philadelphia: W.B. Saunders, 1970: Tonks DB Quality in Clinical Laboratories, Warner-Chilcot Laboratories, Diagnostic Reagents Division, Scarborough, Canada, Westgard JO, Barry PL, Hunt MR, Groth T. Clin Chem 1981;27: Martinello F, Silva E.L. Interferência do ácido ascórbico nas determinações de parâmetros bioquímicos séricos: estudos in vivo e in vitro. Jorn. Bras. Pat. Med. Lab, 2003;39: Basques JC. Especificações da Qualidade Analítica. Labtest Diagnóstica Burtis CA, Ashwood ER. Textbook of Clinical Chemistry, 2a. edição, Philadelphia: W.B. Saunders, 1986: Labtest: Dados de Arquivo. Application procedures are available for various automated systems. The number of testes in automated systems depends of the programmed parameters. Consumer information [Warranty conditions] Labtest Diagnóstica warrants the performance of this product under the specifications until the expiration date shown in the label since the application 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 - Consumer Service sac@labtest.com.br Edition: February, 2012 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 04 English - Ref.: 134

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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.: 134