GLUCOSE Liquiform. 01 English - Ref.: 133. Ref.:133. Insert. Intended use. Methodology. Reagents. Principle Precautions and warnings. Summary.

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1 GLUCOSE Liquiform Insert Ref.:133 Intended use. Enzymatic system for the determination of glucose in blood, cerebrospinal, ascitic, pleural and synovial fluids by kinetic or endpoint method. [Use only for in vitro diagnosis.] Principle. Glucose oxidase catalyses the oxidation of glucose according to the following reaction: Glucose + O + H O Summary. 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. POD 2H O + 4-aminoantipyrine + phenol Antipyrilquinoneimine + 4H O Reagent is presented ready for use and employs enzymatic methodology of high analytical specificity, simple and easy to use in the clinical laboratory. Labtest developed the Glucose Liquiform 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 Liquiform 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. The system allows the determination to be carried out in a fixed-time kinetic method yielding results within only 90 seconds of reaction, and also in endpoint method that provides more agility for certain biochemical analyzers. The method can be used in manual technique and it is easily applicable to semi-automated and automated analyzers capable to accurately measure the absorbance between 490 and 520 nm. Methodology. Reagents Materials needed and not provided GOD - Trinder Reagent 1 - Store at 2-8 ºC. Contains phosphate buffer at 30 mmol/l, ph 7.5, phenol 1 mmol/l, glucose oxidase U/L, peroxidase 0 U/L, 4-aminoantipyrine 290 µ mol/l, sodium azide at 7.5 mmol/l, and surfactants. Calibrator Standard - Store at 2-30 ºC. Contains: glucose at 100 mg / dl and non-toxic biocide. After handling, it is suggested to store and seal to prevent evaporation. The standard stabilizer may precipitate at low temperatures, which does not interfere with its performance. 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. Water bath maintained at constant temperature (37 ºC). Photometer able to accurately measure the absorbance between 490 and 520 nm. Pipettes to measure samples and reagent. 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. 01 English - Ref.: 133

2 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% 4. 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 ºC, 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 Endpoint method. Bilirubin concentrations up to 2.5 mg/dl and hemoglobin up to 200 mg/dl do not produce significant interference. Bilirubin concentrations greater than 2.5 mg/dl produce negative interference. Triglycerides concentrations up to 1000 mg/dl do not produce significant interference when using blank sample. 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. Blank Sample. Hemoglobin Absorbance405 x 601 Hemoglobin absorbance x 467 This procedure is applicable whenever there is positive action for interference. Mix 1.0 ml of NaCl at 150 mmol/l (0.85%) with 0.01 ml of sample. Measure the absorbance at 505 nm, adjusting the zero with deionized or distilled water. Subtract the obtained absorbance from the absorbance of the test, and calculate the concentration. Kinetic method. Bilirubin concentrations up to 2.5 mg/dl, hemoglobin up to 200 mg/dl and triglycerides up to 1000 mg/dl do not produce significant interference. Manual procedure Endpoint method. Sample Standard Reagent 1 Take 3 test tubes, and proceed as follows: Mix vigorously and incubate in a water bath at 37 ºC for 10 minutes. The water level in the bath must be above the reagent level in the test tubes. Determine the absorbance for Unknown and Standard at 505 nm ( ), adjusting the zero with the blank. Unknown color is stable for 30 minutes. The suggested procedure for measurement is suitable for photometers whose minimum volume of solution for measurement is equal to or lower than 1.0 ml. The need to adjust the volume to the photometer used should be checked. Volumes of sample and reagent can be modified proportionately without prejudice to the test performance and the calculation procedure remains unchanged. In case of reduced volumes, it is essential to observe the minimum volume required for photometric reading. Sample volumes lower than 0.01 ml are critical in manual applications and should be used cautiously as they increase the inaccuracy of the measurement. Calculations. See measurement/reportable range. Unknown Absorbance Glucose = x 100 Standard Absorbance Due to the high reproducibility that can be obtained from the methodology, the result can also be obtained by using the calibration factor. Calibration Factor = Blank 100 Standard Absorbance 415 Unknown 0.01 ml 1.0 ml 1.0 ml Standard 0.01 ml 1.0 ml 02 English - Ref.: 133

3 Glucose x = Unknown Absorbance X Calibration Factor Kinetic method. It should be used in all lipemic samples. The procedure uses a two-point kinetic and does not require the blank of the reaction. Temperature control is absolutely essential for the results reproducibility. It is also essential that operations with samples and standards are performed by strictly keeping constant the time interval between mixing of sample or standard with the reagent and the start of photometer measurement. As the reaction time is very short, it is necessary to use a photometer with temperature control at 37 ºC in the cuvette (thermostated cuvette). Adjust the zero of the photometer at 505 nm ( ) with distilled or deionized water. Add 0.01 ml of sample or Standard to 1.0 ml of Reagent 1 preheated to 37 ºC. Mix and immediately start the photometric measurement. Perform a measurement of absorbance at 30 seconds and another measurement at 90 seconds, holding the reaction temperature controlled at 37 ºC. Calculations A ( Unknown or Standard) = Absorbance - Absorbance Unknown A Glucose = x 100 Standard A Calibration. The Standard is traceable to the Standard Reference Material (SRM) 917 of the National Institute of Standards and Technology (NIST). Manual Calibrations Get the calibration factor when using a new batch of reagents or when the internal quality control indicates it. Automated Systems Blank for reagents: water or sodium chloride solution at 150 mmol/l (0.85%); Standards: use protein calibrators. The glucose concentration in the calibrator of Calibra - Labtest line is traceable to NIST SRM 917. Calibration Intervals Calibration of blank when using a new reagent bottle, 2-point calibration when changing the batch; 2-point calibration when the internal quality control indicates it. Measurement/reportable range The measurement result is linear up to 500 mg/dl. When obtaining a value equal to or greater than 500 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 500 mg/dl. Quality 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. Control 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 components of biological variation (BV). 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 0-1 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 x = IS Units (mmol/l) Performance characteristics Recovery studies. Two samples with glucose concentrations equal to 78 and 150 mg/dl were added with different amounts of analyte, obtaining in the endpoint method recovery between 98% and 99%. The mean proportional systematic error obtained at 120 mg/dl is equal to 1.8 mg/dl or 1.5%. Method comparison. The proposed method was compared to a similar method having the following results: Number of samples Range of concentrations Regression equation Correlation coefficient By using the regression equation, the total error (constant and proportional) checked at the decision limit (120 mg/dl) was equal to 1.58 mg/dl or 1.32%. The total error obtained in the same decision level is 4.32%. The results for the comparative study meet the specification for Total Systematic Error of 6.9% for Biological Variation. 12 Comparative Method mg/dl mg/dl Labtest Method Labtest method (mg / dl) = x Comparative English - Ref.: 133

4 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). Precision studies. Precision studies were carried out using samples with mean concentrations equal to 47, 129 and 194 mg/dl. Imprecision - Within 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 1 mg/dl is equal to 6.17%, 4.32% and 4.83%, respectively. The results indicate that the method meets the desired specification for total error ( 6.9%) based on the components of the Biological Variation. Analytical sensitivity. A protein sample containing 44 mg/dl of glucose was used to calculate the detection limit for the assay, being found a value equal to 1.77 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 634 and 625 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 95 and 99%. Notes 1. The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results. 2. N Imprecision - Run-to-run Sample 1 Sample 2 Sample 3 N Mean Mean SD SD %CV %CV 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 <0.1mg/L. References 1. Bergmeyer HU. Methods of Enzimatic Analysis, 3a. edição, Vol VI, Deerfield Beach:VCH, 1986: Blaedel WJ, Uhl JM. Clin Chem 1975;21: Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests, 1a. edição, Washington: AACC Press, Sociedad Española de Bioquímica Clínica y Patología Molecular, Base de Datos de Variación Biológica. Disponível em:< ticle/ar ticleview/330/1/170> (acesso em 08/2006). 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 Control 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: Data on file. Presentation Product Reference Contents Glucose Liquiform Consumer information [Warranty conditions] 133-1/ /500 1 x 500 ml 1 x 5 ml 2 x 500 ml 1 x 5 ml 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 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. 04 English - Ref.: 133

5 Labtest Diagnóstica S.A. CNPJ: / Av. Paulo Ferreira da Costa, Vista Alegre - CEP Lagoa Santa. Minas Gerais Brasil - Consumer Service sac@labtest.com.br Revision: December, 2011 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 05 English - Ref.: 133

6 06 English - Ref.: 133