1. Reagent Store at 2-8 ºC.

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1 LDL Insert Ref.:146 Intended use. System for direct quantitative determination of low density lipoprotein (LDL) in serum or plasma samples. Professional use. [For in vitro diagnostic use.] Test principle. In the first step of the reaction, the polyanion and the non-ionic surfactant inhibit the reaction of the high density lipoprotein (HDL), the very low density lipoprotein (VLDL) and the chylomicrons (CM) with the cholesterol oxidase (CHOD) and cholesterol esterase (CHER) enzymes. In the second step, in the presence of non-ionic surfactant, the enzymes CHOD and CHER react specifically with LDL in sample, yielding H O, 2 2 which is detected by the Trinder reaction. The color intensity is directly proportional to the concentration of HDL cholesterol in sample. Polyanion LDL+(HDL+VLDL+CM) HDL+LDL+VLDL+CM Polyanion/Non-ionic surfactant Non-ionic surfactant CHOD + CHER LDL Cholest-4-en-one + fatty acid + H O Non-ionic surfactant Peroxidase 2H O + 4-Aminoantipirine + EMSE Quinoneimine + 5H O Summary. The procedure to determine the LDL cholesterol concentration that combines ultracentrifugation and chemical precipitation, based on the reference method from the Centers for Disease Control and Prevention (CDC) is time consuming and has little use in 1 clinical laboratories. The LDL system is a liquid homogeneous method to measure LDL cholesterol directly in a simple and easy process. The total error obtained meet the performance requirements established by the National 1 Cholesterol Education Program (NCEP - USA) since The specificity is obtained by selective action of a surfactant, which allows cholesterol consumption in non-ldl particles. Triglycerides up to 1500 mg/dl, no conjugated bilirubin up to 30 mg/dl, conjugated bilirubin up to 30 mg/dl, hemoglobin up to 500 mg/dl, and ascorbic acid up to 50 mg/dl do not interfere significantly in the reaction. The method flexibility makes it easily applicable to automated systems capable of reading end-point reactions with two reagents. 2 2 Methodology. Selective surfactant Reagents 1. Reagent Store at 2-8 ºC. Ready to use. Contains buffer 20.1 mm ph 7.0; polyanion 0.6 g/l; 4-aminoantipyrine mmol/l; and sodium azide 0,1% Reagent 2 - Store at 2-8 ºC. Ready to use. Contains buffer 20.1 mm ph 7.0; cholesterol oxidase U/mL, cholesterol esterase U/mL, peroxidase U/mL; EMSE 0.72 a 2.84 mmol/l; non-ionic surfactant 8.5 g/l e sodium azide 01% Calibrator -Store at 2-8 º C. Lyophilized reagent. Check the calibrator concentration on the bottle label. Preparation containing human LDL cholesterol; buffer 50 mm ph7.4; sodium chloride <150 mm and stabilizers. Unopened reagents, when stored at indicated temperature, are stable up to the expiration date shown on the label. When kept in the equipment's tray with cooling system, reagents 1 and 2 are stable for 5 weeks. This stability period may change according to the equipment's characteristics and maintenance conditions. Therefore, it is recommended that the quality control be used to monitor the reagent's performance. After opened, the reagents are stable up to the expiration date shown on the label, but only when stored in a tightly closed vial at 2-8 º C and without microbial or chemical contamination. Precautions and warnings The usual security cares should be applied to the reagent handling. They must not be pipetted by mouth aspiration. The calibrator contains human blood derivatives and was tested for the presence of HBsAg and anti-hcv and anti-hiv antibodies with approved tests, yielding negative results for all of them. 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. The calibrator 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. 01 English - Ref.: 146

2 Materials required not provided 1. Analyzer capable of measuring absorbance accurately at nm. 2. Incubator or water bath kept constantly at 37 º C. 3. Pipets to measure samples, calibrator, controls and reagents. 4. Timer. Sample Use serum or plasma (EDTA, lithium heparin). Serum or plasma samples must not be kept at room temperature (15-30 º C) for more than 14 hours. If it is not possible to perform tests within 14 hours after sample collection, samples can be stored at 2-8 º C for up to 5 days and at -20º C for up to 30 days. If samples must be stored for longer periods, they must be kept at temperatures -70 º C. Avoid repeated freeze and thaw cycles. Thawed samples must be homogenized thoroughly prior to being tested. Do not use vortexing system or similar equipment. Do not use samples with signs off microbial contamination. 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. Since no known test method can offer complete assurance that human blood samples will not transmit infectious diseases, all blood samples should be considered potentially infectious and handled accordingly. Disposal of all biological waste material should be in accordance with local guidelines. Procedure - automated systems Parameters for automated analyzers Parameter Reaction Type Wavelength (Primary) Wavelength (Secondary) Temperature Calibration Calibration Model Sample Volume* R1 Volume* R2 Volume* Reading 1 (Abs 1) Addition of R2 Reading 2 (Abs 2) *The sample and reagent volumes can be modified proportionally without any loss in test performance. In case of volume reduction it is crucial to observe the minimal necessary volume for photometric reading. Reaction model Application End point 600 nm 700 nm 37 ºC 2 Points Point 1: Deionized water or sodium chloridesolution 150mmol/L(0.85%) Point 2:CalibratorRef146.3 Linear 3.5 L 210 L 70 L After 300 seconds of incubation at 37 º C of R1 + sample After 300 seconds of incubation of R1 + sample After 300 seconds of incubation at 37 º C of R1 + sample + R2 Interference Unconjugated non-conjugated bilirubin up to 30 mg/dl, conjugated bilirubin up to 30 mg/dl, hemoglobin up to 500 mg/dl, ascorbic acid up to 50 mg/dl and triglycerides up to 1500 mg/dl do not interfere significantly in the reaction. Preparing the calibrator. Add 1.0 ml of deionized water (see Note 2) to the Calibrator vial. Let it stand for 5 minutes Mix the vial by gentle inversion, avoiding formation of foam. The reconstituted calibrator is stable for 14 days at 2-8 º C in a tube proper for liquid storage, which avoids solvent evaporation. Calibration. The calibrator is traceable to the CDC (Centers for Desease Control) Method though a traceability chain. Calibration interval. 2-point calibration when the lot is changed or when the quality control indicates so. R1: 210 L Sample: 3.5 L Calculation. See Operating Interval A=A2-A1 R2: 70 L 0 min 5 min 37 ºC 37 ºC A1 (600/700 nm) 10 min A2 (600/700 nm) Concentration of LDL Asample - Ablank Calibrator cholesterol in sample : X (mg/dl) A calibrator - A blank 02 English - Ref.: 146

3 Operating interval. The reaction is linear for LDL concentrations between 1.00 and 600 mg/dl. For samples with LDL concentration above 600 mg/dl, dilute the sample with NaCl 150 mmol/l (0.85%), perform a new test and multiply the result obtained by the dilution factor. 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. Control materials should be used for measurement imprecision monitoring and determination of calibration deviation. It is recommended to use the products Qualitrol - Labtest as internal quality control. Expected values. These values substitute the reference values and were determined from statistically-treated epidemiologic data, which correlate cholesterol concentration and prevalence of ischemic coronary disease (ICD) Performance characteristics Method comparison. The proposed method was compared against a similar method for direct determination of LDL, and the following results were obtained: Samples Mean (mg/dl) Regression equation Correlation coefficient Comparative Method Labtest Method LDL= 1.002*(Comparative Method) mg/dl Using the regression equation, the systematic error (bias) was equal to 0.32% and 0.26 for samples with concentrations of 60.3 mg/dl and mg/dl, respectively ATP III classification - Adults Total Cholesterol (mg/dl) < LDL Cholesterol (mg/dl) < HDL Cholesterol (mg/dl) < Borderline Borderline desirable Borderline elevated Very elevated Low (desirable) Imprecision. The imprecision studies were performed using two native samples. Imprecision - Within Run Mean n (mg/dl) Sample 1 Sample 2 Imprecision - Run-to-Run Sample 1 Sample 2 SD (%) CV n Mean (mg/dl) SD (%) CV Classificação ATP III - Children and Adolescents 3 Total Cholesterol (mg/dl) - Age: 2 to 19 years < Borderline 200 LDL Cholesterol (mg/dl) - Age: 2 to 19 years < Borderline 130 HDL Cholesterol (mg/dl) - Age: <10 years 40 The imprecision found meets the NCEP - USA specification, which is 4.0% The total error (random error + systematic error) estimated are 1.24% and 1.48% for the decision levels of 60.3 and mg/dl, respectively. The results indicate that the method meets the specification for Total Error ( 12.0%) from the NCEP - USA. Analytical sensitivity. Detection limit of the method: 1.0 mg/dl. Effect of matrix dilution. A sample with LDL concentration equal to mg/dl was used to evaluate the system response to matrix dilution using NaCl 150 mmol/l (0.85%) solution. Using dilution factors ranging from 1.5 to 3, the mean recovery found was 98.2%, which corresponds to a mean systematic error of 1.8% HDL Cholesterol (mg/dl) - Age: 10 to 19 years 35 mg/dl x = mmol/l LDL cholesterol 03 English - Ref.: 146

4 Notes 1. The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results. 2. 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. 3. Find a review of physiopathological sources and drugs which interfere in results and methodology at Young, DS. Effects of Drugs on Clinical Laboratory Tests, 5rd edition, Washington: AACC Press, References 1. Bachorik PS, Ross JW. Clin Chem 1995;41: Executive Summary of the Third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285: Presentation Product Reference Content LDL The number of tests in automated systems depends on the programmed parameters. 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 Edition: July, 2016 Ref.: / X 30 ml 2 1 X 10 ml 1 X 1.0 ml CAL customerservice@labtest.com.br Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 3. Leite PF, Martinez TLR, Halpern A, Cendoroglo MS, Novazzi JP, Fonseca FAH, Dias JCA. Risco cardiovascular: fatores metabólicos e nutricionais: diagnóstico e tratamento. São Paulo: Loyola, p Labtest: Data on file 04 English - Ref.: 146

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