LDL LD. 01 English - Ref.: 129. Ref.:129. Insert. Intended use. Methodology. Reagents. Test principle. Summary. Precautions and warnings

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1 LDL LD Insert Ref.:129 Intended use. System for direct quantitative determination of low density lipoprotein (LDL) in serum or plasma samples. [For in vitro diagnostic use.] Test principle. The method is based on a modification of classic coprecipitation methods using optimized amounts of polyvinyl sulfonic acid (PVS) and polyethylene glycol methyl ether (PEGME) associated to selective surfactants. On the first step of the reaction, LDL, VLDL, and chylomicrons (CM) react with PVS and PEGME, which makes them inaccessible to cholesterol oxidase (CHOD) and cholesterol esterase (CHER), while HDL reacts with those two enzymes. After the addition of reagent 2, which contains specific surfactants that release LDL from the PVS/PEGME complex, LDL reacts with CHOD and CHER, yielding H2O 2, which is detected by the Trinder reaction. The color intensity is directly proportional to the concentration of LDL cholesterol in sample PVS / PEGME HDL + LDL + VLDL + CM HDL+ (LDL + VLDL + CM). PVS/PEGME Summary. HDL + CHOD + CHER Fatty acid + H2O2 Specific surfactants (LDL + VLDL + LDL + CM). PVS/PEGME LDL + (VLDL + CM). PVS/PEGME LDL + CHOD + CHER Fatty acid + H2O2 2H2O AA + TODB Quinoneimine + 5H2O 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 clinical laboratories. The LDL LD system is a liquid homogeneous method to measure LDL cholesterol directly in a simple and easy process, with precision and accuracy levels that meet the performance requirements established by the National Cholesterol Education Program (NCEP - USA) since The specificity is obtained by selective action of a surfactant in reagent 2, which allows cholesterol consumption in non-ldl particles. Triglycerides up to 1000 mg/dl, bilirubin up to 40 mg/dl, conjugated bilirubin up to 30 mg/dl, hemoglobin up to 1000 mg/dl, and ascorbic acid up to 10 mm 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 and measuring absorbance at 600 nm. Methodology. Reagents CAL - Selective surfactant Reagent 1 - Store at 2-8 ºC. Ready to use. Contains MES buffer ph 6.5 <500 mm; polyvinyl sulfonic acid <50 g/l; polyethylene glycol methyl ether <50 g/l; magnesium chloride <50 mm; cholesterol esterase <100 U/L; cholesterol oxidase <100 U/L; 4-aminoantipyrine <50 mm; peroxidase <100 U/L; preservative; and surfactant. Reagent 2 - Store at 2-8 ºC. Ready to use. Contains MES buffer ph 6.5 <500 mm; TODB N,N-bis (4-sulfobutyl)-3-methylaniline <50 mm; EDTA <10 mm; preservative; and surfactant. Calibrator 1 - Lyophilized reagent. Store at 2-8 ºC. Check the calibrator concentration on the bottle label. Preparation containing human LDL cholesterol and sodium azide 0.1%. 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 60 days. 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 ºC 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.: 129

2 Materials required not provided 1. Analyzer capable of measuring absorbance accurately at nm Incubator or water bath kept constantly at 37 ºC. Pipets to measure samples, calibrator, controls and reagents. Timer. Specimen collection and preparation Use serum or plasma (EDTA, citrate). Serum or plasma samples must not be kept at room temperature (15-30 ºC) 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 ºC for up to 5 days and at -20 ºC for up to 30 days. If samples must be stored for longer periods, they must be kept at temperatures -70 ºC. 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. Procedure - automated systems * 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. Parameter Reaction Type Wavelength Temperature Calibration Calibration Model Sample Volume* R1 Volume* R2 Volume* Reading 1 (Abs 1) Addition of R2 Reading 2 (Abs 2) Application End point 600 nm 37 ºC 2 Points Point 1: Deionized water or sodium chloride solution 150 mmol/l (0.85%) Point 2: Calibrator Ref Linear 3 µ L 225 µ L 75 µ L After 300 seconds of incubation of R1 + sample at 37 ºC After 300 seconds of incubation of R1 + sample After 300 seconds of incubation of R1 + sample + R2 at 37 ºC 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. Reaction model R1: 225 µ L Sample: 3 µ L R2: 75 µ L Interference Unconjugated bilirubin up to 40 mg/dl, conjugated bilirubin up to 30 mg/dl, hemoglobin up to 1000 mg/dl, ascorbic acid up to 10 mm and triglycerides up to 1000 mg/dl do not interfere significantly in the reaction. 0 min 5 min 37 ºC 37 ºC 10 min 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 ºC in a tube proper for liquid storage, which avoids solvent evaporation. Calibration The calibrator is traceable to the Standard Reference Material (SRM) 1951 from the National Institute of Standards and Technology (NIST). Calibration interval 2-point calibration when the lot is changed or when the quality control indicates so. Calculation. A = A2 - A1 See Operating Interval Concentration of LDL cholesterol in sample = A sample - A blank A calibrator - A blank Operating interval. A1 (600 nm) x calibrator (mg/dl) A2 (600 nm) The reaction is linear for LDL concentrations up to 250 mg/dl. For samples with LDL concentration above 250 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. 02 English - Ref.: 129

3 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 Labtest method = 1.08 x Comparative mg/dl 0.98 ATP III classification - Adults 2 Total Cholesterol (mg/dl) < LDL Cholesterol (mg/dl) < Borderline Borderline desirable Borderline elevated Very elevated Using the regression equation, the systematic error (bias) was equal to 8.63%, 8.41%, and 8.28% for samples with concentrations of 95 mg/dl, 146 mg/dl, and 210 mg/dl, respectively Imprecision. native samples. Imprecision - Within Run Sample 1 Sample 2 Sample 3 The imprecision studies were performed using three N Mean (mg/dl) SD (%) CV HDL Cholesterol (mg/dl) <40 60 Classificação ATP III - Children and Adolescents 3 Low (desirable) Total Cholesterol (mg/dl) - Age: 2 to 19 years < Borderline 200 LDL Cholesterol (mg/dl) - Age: 2 to 19 years < Borderline 130 Imprecision - Run-to-Run Sample 1 Sample 2 Sample 3 N Mean (mg/dl) The imprecision found meets the NCEP - USA specification, which is 4.0% The total error (random error + systematic error) estimated for the decision levels of 95 mg/dl, 146 mg/dl, and 210 mg/dl are 11.27%, 10.88%, and 10.59%, respectively. The results indicate that the method meets the specification for Total Error ( 12.0%) from the NCEP - USA. SD (%) CV HDL Cholesterol (mg/dl) - Age: <10 years 40 Analytical sensitivity. A sample without LDL was used to calculate the detection limit of the method, and a value of 1.64 mg/dl was found, which is equivalent to 12 assays plus three standard deviations. HDL Cholesterol (mg/dl) - Age: 10 to 19 years 35 mg/dl x = mmol/l LDL cholesterol Effect of matrix dilution. A sample with LDL concentration equal to 250 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 2 to 5, the mean recovery found was 95.38%, which corresponds to a mean systematic error of 4.62% 03 English - Ref.: 129

4 Notes 1. The material cleaning and drying are fundamental factors to the reagent stability and to obtain correct results. 2. The water in the laboratory to prepare reagents and use in the measurements must have resistivity 1 megaohm, or conductivity 1 microsiemens and silicates concentration must be <0.1mg/L (Type II reagent water). The water for washing must be Type III, having resistivity 0.1 megaohms or conductivity 10 microsiemens. For the final washing, use Type II reagent 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, 3 edition, Washington: AACC Press, References rd Presentation Product Reference Content LDL LD The number of tests in automated systems depends on the programmed parameters. Consumer information [Warranty conditions] 129-1/ X 60 ml 2 1 X 20 ml 1 X 1.0 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 CAL 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: 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 Labtest Diagnóstica S.A. CNPJ: / Av. Paulo Ferreira da Costa, Vista Alegre - CEP Lagoa Santa. Minas Gerais Brasil - Consumer Service Edition: October, 2011 Ref.: sac@labtest.com.br Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 04 English - Ref.: 129

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