HDL LE. 01 Inglês - Ref.: 98. Ref.:98. Insert. Methodology. Intended use. Reagents. Test principle Precautions and warnings. Summary.

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1 HDL LE Insert Ref.:98 Intended use. For the quantitative determination of high-density lipoprotein cholesterol (HDL-C) in serum and plasma. Test principle. This method is based on accelerating the reaction of cholesterol oxidase (CO) with non-hdl unesterified cholesterol and dissolving HDL selectively using a specific detergent. In the first reagent, non-hdl unesterified cholesterol is subject to an enzyme reaction and the peroxide generated is consumed by a peroxidase reaction with DSBmT yielding a colorless product. The second reagent consists of a detergent capable of solubilizing HDL specifically, cholesterol esterase (CE) and chromagenic coupler to develop color for the quantitative determination of HDL-C. This may be referred to as the Accelerator Selective Detergent methodology. HDL, LDL, VLDL, Chylomicrons HDL HDL Cholesterol H O + DSBmT + 4-AAP 2 2 Summary. Accelerator + CO DSBmT + Peroxidase HDL Specific Detergent Cholesterol esterase Cholesterol oxidase Peroxidase on-reactive LDL + HDL VLDL, Chylomicrons HDL Disrupted Cholesterone + H O Color Development Due to importance of the accurate measurement of the HDL-C, is desirable to use a reference method. The reference method for the quantization of HDL-C combines ultracentrifugation and chemical precipitation to separate HDL from other lipoproteins, followed by cholesterol measurement by Abell-Kendall analysis. This method is too 6 time consuming and labor intensive for use in routine analysis. The HDL LE system is a homogeneous method for directly measuring HDL-C levels, and consists in a simple process and well defined that transmits confidence in the methodology accuracy. The method has an excellent correlation with MDC method and presents accuracy and precision characteristics that significantly exceed the performance parameters proposed by the ational Education Cholesterol Program (CEP) since 998. The desired specificity is obtained with the action of a unique detergent present in Reagent 2, whose properties allow dissolving only the HDL-C. The system flexibility allow it to be easily applied to most automatic and semi-automatic equipments which are able to process a reaction with 2 reagents and measure an end point reaction at 600 nm. 2 2 Methodology. Reagents Storage and stability. Deterioration. Accelerator Selective Detergent. Reagent - Store at 2-8 ºC. Reagent label bears expiration date. Buffer ph 6.0; cholesterol oxidase <000 U/L; peroxidase <300 U/L; DSBmT < mmol/l; accelerator < mmol/l and sodium azide <0.06%. Reagent 2 - Store at 2-8 ºC. Reagent label bears expiration date. Buffer ph 6.0; cholesterol esterase <500 U/L; 4-aminoantipyrine <mmol/l; ascorbic acid oxidase <3000 U/L; detergent <2% and sodium azide <0.06%. Calibrator - Store at 2-8 ºC. Calibrator label bears expiration date and concentration. Lyophilized reagent. Preparation containing human HDL cholesterol, buffer 50 mm, sodium chloride >50 nm and stabilizers <5%. Precautions and warnings For in vitro diagnostic use. Disposal of all waste material should be in accordance with local guidelines. The usual security cares should be applied on the reagent handling. The reagents contain sodium azide as preservative. Avoid ingestion. In case of eyes contact, immediately flush eyes with plenty of water and get medical assistance. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide accumulation. The Calibrator contains human blood derivates and it was tested to the presence of HBsAg and, HCV and HIV antibodies yielding negative results. However, no known test method can offer complete assurance that human blood samples will not transmit infectious diseases. Therefore, all blood derivatives should be considered potentially infectious. Unopened reagents, when stored at indicated temperature, are stable up to expiration date shown on the label. reagents stability. Microbial or chemical contamination may decrease Specimen collection and preparation HDL-C is reportedly stable in serum for about 7 days at 2-8 ºC and 30 days at -20 ºC. 0 Inglês - Ref.: 98

2 Use serum or plasma (heparin and EDTA). Samples with citrate or oxalate should not be used because they yield false decreased results. Interference Bilirubin up to 60 mg/dl, hemoglobin up to 000 mg/dl, ascorbic acid up to 00 mg/dl, triglycerides up to 800 mg/dl and gammaglobulin up to 5000 mg/dl do not interfere significantly. Sample presenting interferences values over those cited above should be diluted using 0,85% acl (50 mmol/l) before measuring. Multiply the result by the dilution factor. Materials required not provided Timer. A constant temperature water bath (37 ºC). Photometer capable of measuring absorbance at 600 nm. Pipettes to measure reagents and samples. Preparing the calibrator. Add.0 ml of reagent water to the Calibrator bottle. Rest it for 5 minutes. Mix gently by inversion avoiding foam formation. It is stable 7 days at 2-8 ºC and 30 days at -20 ºC if the bottle is tightly close. Avoid repeated freezing and melting, we suggest to separate the standard in small amounts before freezing. Manual procedure Set up two tubes and proceed as follows: Calibration frequency Two point calibration after reagent lot change; Two point calibration when the internal quality control indicates. Quality control. For quality control use Qualitrol H Level and Level 2 or other suitable control material. The limits and control interval must be adapted to the laboratory requirements. Each laboratory should establish corrective actions to be taken if values fall outside the control limits. Calculations. See linearity. The first absorbance measurement (A ) must be fixed to the final volume of reaction, obtaining Ac before the HDL concentration calculation. A c = A x 0.75 HDL cholesterol = Test (A2 - A c) Calibrator (A2 - A c) x CC CC: Calibrator concentration. Due the high reproducibility that this methodology offers, the factor method may be applied. Factor = CC Calibrator (A2 - A c) HDL cholesterol = Test (A2 - A c) x factor Sample Calibrador Reagent Test Calibrator 0.0 ml 0.0 ml 0.75 ml 0.75 ml Measurement/reportable range The reaction is linear from 2.5 to 200 mg/dl. If HDL-C concentration exceeds 200 mg/dl, the sample must be diluted with 0,85% acl. Multiply the result by the appropriate dilution factor. Mix and incubate in a water bath at 37 ºC during 5 minutes. The water level in the water bath must be higher than the level of reagents in the tubes. Measure the absorbance A of the test and Calibrator against water at 600 nm. Add 0.25 ml of the Reagent 2 in each tube. Keep in the water bath at 37 ºC during 5 minutes. Measure the absorbance A2 of the test and calibrator at 600 nm against water. If the photometer is able to bichromatic readings, use a primary filter with emission at 600 nm and a secondary filter with emission ranging from 660 to 700 nm. Calibration Expected values. The following values substitute the reference values and were determined from epidemiologic data statistically analyzed that relate cholesterol levels to the Ischemic Coronary Disease (ICD) prevalence. Adults 2 Total cholesterol Expected <200 Increased threshold Increased 240 Manual calibrations Perform a new calibration after reagent lot change or when the internal quality control indicates. The Calibrator is traceable to IST SRM 95. Automatic Systems Blank of reagents: water or 0.85% acl; Standard: Calibrator LDL cholesterol Excellent Excellent threshold Increased threshold Increased Very increased < Inglês - Ref.: 98

3 HDL cholesterol Low <40 Increased (expected) 60 Children and adolescents 9 Total cholesterol 2-9 years old Expected <70 Threshold Increased 200 LDL cholesterol 2-9 years old Expected <0 Threshold 0-29 Increased 30 The estimated total error (random + systematic) in a decision point of 40 mg/dl is 4.06 mg/dl or 0.2%. Imprecision - Within run Sample Sample 2 Sample Reference Method (RM) Mean SD HDL LE Range Mean Regression Analysis HDL LE =.0 x ( RM) mg/ dl Correlation Coefficient %CV HDL cholesterol < 0 years old Expected 0-9 years old Expected Performance characteristics Imprecision - Run-to-run Sample Sample 2 Sample Mean SD %CV.2.4. Accuracy. Accuracy of the HDL LE method was verified by comparison to the Designated Comparison Method (DCM) for HDL producing the following results: Designated Comparison Method (DCM) Range Mean Regression Analysis HDL LE = 0. 99* ( DCM) mg/ dl Correlation Coefficient The estimated total error (random + systematic) in a decision point of 40 mg/dl is 4.39 mg/dl or.0%. Other Performance Studies (Specificity). HDL LE In a study comparing the HDL LE method to the Reference Method (RM) for HDL cholesterol, 4 patient specimens with triglyceride levels greater than the 400 mg/dl ( mg/dl) were analyzed producing the following results: Effects of matrix dilution. A sample with HDL-C value equal to 200 mg/l was used for evaluating the system response on dilution with 50 mmol/l acl (0.85%) with a deviation from the linear line of less than or equal to 5%. Analytical sensitivity. Detection limit: 0.57 mg/dl. The detection limit represents the lowest measurable HDL-C concentration that can be distinguished from zero. It is calculated as two standard deviations of 20 replicates of one sample without HDL-C. otes. The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results. 2. The clinical laboratory aims to provide accurate and precise results. The use of water of poor quality is a potential cause of analytical errors. The water used in the laboratory must have the appropriate quality for each application. Thus, to prepare reagents, using the measurements and for use in final rinse of the glass, must have resistivity megaohm.cm, or conductivity microsiems/cm and silicates concentration must be <0,mg/L. When the deionized column is saturated with its capacity, various ions, silicates and substances with high oxidation or reduction that deteriorate the reagents in a few days or even hours are release, changing the results so unpredictable. Thus, it is vital to establish a program to control water quality. 03 Inglês - Ref.: 98

4 3. Several factors alter the results obtained with the calibrator. Among these factors are the dissolution, homogenization, water or glasses contaminants, inappropriate control of temperature or technical errors associated to equipment or the reagent systems. It is suggested to follow the Good Practices in Clinical Laboratories and the verification of the equipment and reagents instructions, related to the procedures limitations. 4. The calibrator value was established using HDL LE (Ref. 98) procedures and reagent according to the described use instructions. Due the Matrix effect, the calibration may be not appropriated if it is used reagents from other manufacturer. References. Bachorik PS, Ross JW. Clin Chem 995;4: Executive Summary of the Third Report of the ational Cholesterol Education Program (CEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 200;285: Kimberly MM, Leary ET, Waymack PP. Clin Chem 999;45: Pesce AJ, Kaplan LA. Methods in Clinical Chemistry, St. Louis: The C.V. Mosby Co., 987: Virella MFL, Stone P, Ellis S, Colwell G. Clin Chem 977;23: Warnick RG, Wood PD. Clin Chem 995; 4: Westgard JO, Barry PL, Hunt MR, Groth T. Clin Chem 98; 27: Labtest: Data on file. 9. Leite PF, Martinez TLR, Halpern A, Cendoroglo MS, ovazzi JP, Fonseca FAH, Dias JCA. Risco cardiovascular: fatores metabólicos e nutricionais: diagnóstico e tratamento. São Paulo: Loyola, 994. p.56. Presentation Product HDL LE Application procedures using HDL LE are available for various automated systems. The number of tests in automated systems depends on 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. CPJ: / Av. Paulo Ferreira da Costa, Vista Alegre - CEP Lagoa Santa. Minas Gerais Brasil - Consumer Service Reference sac@labtest.com.br Contents X 60 ml X 20 ml X.0 ml X 240 ml X 80 ml X.0 ml 3 X 2000 ml X 2000 ml X.0 ml Review: March, 203 Ref.: 2803 Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 04 Inglês - Ref.: 98

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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 úmero do lote Denominación de lote Batch code Consultar instruções de uso Consultar instrucciones de uso Consult instructions for use e úmero do catálogo úmero de catálogo Catalog umber e negativo negativo egative 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 Ref.: Inglês - Ref.: 98