LDL Liquiform. 01 English - Ref.: 111. Ref.:111. Insert. Intended use. Test principle. Methodology. Reagents Precautions and warnings

Transcription

1 LDL Liquiform Insert Ref.:111 Intended use. System for direct homogenous determination of LDL cholesterol in serum and plasma Test principle. On the first stage of the reaction, a specific surfactant present in Reagent 1 solubilizes the chylomicrons, high-density lipoproteins (HDL), and very low-density lipoproteins (VLDL) in the sample. The solubilized cholesterol is entirely consumed by Cholesterol Esterase (CE) and Cholesterol Oxidase (CO) in a reaction that does not produce color. On the second stage, a surfactant in Reagent 2 solubilizes LDL, which is hydrolyzed by Cholesterol Esterase, yielding free cholesterol and fatty acids. Free cholesterol is oxidized by Cholesterol Oxidase to cholest-4-enone and hydrogen peroxide. Next, a coupling reaction between hydrogen peroxide, 4-aminoantipyrine and sodium disulfobutylmetatoluidine (DSBmT) is catalyzed by peroxidase, yielding a quinoneimine which has an absorbance peak at 546 nm. The resulting color intensity is directly proportional to the concentration of LDL cholesterol in the sample. First Stage CE + CO LDL + HDL + VLDL + Chylomicron + Surfactant 1 LDL + Colorless Product Second Stage Summary. LDL + Surfactant 2 CE Cholesterol + Fatty Acids CO Cholesterol + O2 Cholest-4-en-one + H2O2 Peroxidase 2 H2O 2 + DSBmT + 4-Aminoantipyrine Quinoneimine + 4 H2O The procedure for determining LDL cholesterol which combines ultracentrifugation and chemical precipitation, based on the reference method (RM) from Centers for Disease and Prevention (CDC) is time-consuming, operation-intensive, and has limited application on clinical laboratories. The LDL Liquiform system from Labtest is a liquid homogenous method for direct measurement of LDL cholesterol with a procedure that is simple and easy to perform. It is substantially equivalent to the reference method and exhibits precision and accuracy that are superior to the performance requirements established by the National Cholesterol Education Program (NCEP), in effect since The LDL Liquiform system is certified by the Cholesterol Reference Method Laboratory Network (CRMLN). The CRMLN certification program uses reference methods or indicated comparative methods that are rigorously standardized against CDC reference methods. The method specificity is achieved through the selective action of the surfactant in Reagent 1, which makes the non-ldl cholesterol consumption possible. Concentrations of triglycerides up to 1293 mg/dl, bilirubin up to 20 mg/dl, hemoglobin up to 500 mg/dl, ascorbic acid up to 50 mg/dl, and gamma globulins up to 5000 mg/dl do not interfere significantly in the reaction. The method flexibility makes the product easily applicable to automated analyzers capable of processing end-point reactions with two reagents. Methodology. Reagents 1. - Selective Surfactant. 1 Reagent 1 - Store at 2-8 ºC. Do not freeze. Reagent label bears expiration date. Contains buffer ph 6.3; cholesterol esterase <1500 U/L; cholesterol oxidase <1500 U/L; peroxidase <1300 U/L; 4-aminoantipyrine <0.1%; ascorbate oxidase <3000U/L; and surfactant <1.0% Reagent 2 - Store at 2-8 ºC. Do not freeze. Reagent label bears expiration date. Contains buffer ph 6.3; surfactant <1.0%; and DSBmT <1.0 mm Store at 2-8 ºC. Lyophilized reagent. Reagent label bears the calibrator LDL concentration and expiration date. After reconstitution, it is stable for two weeks at 2-8 ºC. Contains human LDL cholesterol and sodium azide 0.1%. 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 to the reagent handling. The reagents must be protected from light exposure. The 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 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.: 111

2 Triglycerides concentrations up to 1293 mg/dl do not interfere in LDL cholesterol results. Samples with triglycerides values >1293 mg/dl must not be diluted. Storage and stability. The Reagents 1 and 2, when stored at equipament refrigerated board, are estable up to 4 weeks. This stability time can be modified due to equipament's characteristics and maintence conditions. Therefore, its suggested to use internal quality control to monitor reagents performance. The reagents after oppened, when stored tightly closed at 2-8 ºC and when not submitted to chemical and microbial contamination, are stable up to the expiration date shown in the label. In order to avoid evaporation of the, keep the bottle tightly closed. Deterioration. reagent stability. Microbial or chemical contamination may decrease Use serum or plasma (lithium heparin and EDTA). Samples with citrate or oxalate must not be used, for they yield falsely decreased results. Serum or plasma samples may be kept at ºC for up to 14 hours. When the measurement is not to be performed within 14 hours after blood collection, samples can be stored at 2-8 ºC for 5 days and for 30 days at - 20 ºC. Samples must be stored at -80 ºC when it is necessary to preserve them for longer periods. Freeze and thaw cycles should be avoided. Thawed samples must be homogenized prior to measuring. Do not use vortex or other homogenizing device. Do not use samples with signs of microbial contamination. 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. Triglycerides up to 1293 mg/dl, bilirubin up to 20 mg/dl, hemoglobin up to 500 mg/dl, ascorbic acid up to 50 mg/dl, and gamma globulins up to 5000 mg/dl do not interfere significantly in the reaction. Samples with triglyceries levels above 1293 mg/dl must not be diluted. Samples with bilirubin, hemoglobin, ascorbic acid, or gamma globulins levels above the ones previously referred must be diluted with NaCl 150 mmol/l (0.85%) before performing the tests, and the results obtained must be multiplied by the dilution factor. Materials required not provided Specimen collection and preparation Interference A constant temperature water bath (37 ºC). Photometer capable of measuring absorbance at nm. Pipets to measure reagents and samples. Timer. Preparing the calibrator. Add 1.0 ml of deionized or distilled water to the vial. Let it stand for 5 minutes. Mix gently by inversion, avoiding the formation of foam. It is stable for 2 weeks at 2-8 ºC, in a tightly shut vial to avoid evaporation. Manual procedure This procedure is not applicable to semi-automatic analyzers that use flow cells exclusively. Set up two tubes and proceed as follows: Sample Reagent 1 Mix and incubate in a water bath at 37 ºC for 5 minutes. Measure the A absorbance of the Unknown and against water at 546 nm ( ). Reagent 2 Mix and incubate at 37 ºC for 5 minutes. Measure the A2 absorbance of the Unknown and against water at 546 nm ( ). The color is stable for 15 minutes. Calibration. Quality control. Calculations. The calibrator is traceable to CDC reference method. Manual calibrations Perform a new calibration after reagent lot change or when the internal quality control indicates. Automatic Systems Blank of reagents: water or 0.85% NaCl; Standards: use the from the product. Calibration frequency Two point calibration after reagent lot change; Two point calibration when the internal quality control indicates. For quality control use Qualitrol H Level 1 and Qualitrol H 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. The first absorbance measurement (A ) must be corrected to the final volume of the reaction (A LDL concentration. A = A x cor 1 LDL (mg/dl) = Unknown 0.01 ml 0.75 ml Unknown (A - A ) (A - A ) x C 1cor 0.01 ml 0.75 ml 0.25 ml 0.25 ml 1 ) before calculating the 1 02 English - Ref.: 111

3 Due to the high reproducibility of the method, the LDL concentration can be calculated using the factor method. FACTOR = C (A - A ) LDL (mg/dl) = Unknown (A - A ) x Factor Analytical measurement range. The rate of the reaction is linear between 6.6 and 992 mg/dl. Samples with LDL concentrations higher than 992 mg/dl must be diluted with NaCl 150 mmol/l (0.85%). Perform a new LDL test with the diluted sample and multiply the result by the dilution factor. Expected range. The following reference values were determined using statistically treated epidemiologic data, and they correlate cholesterol levels to coronary artery disease (CAD) prevalence. ATP III Cholesterol Guidelines - Total Cholesterol, LDL, and HDL (mg/dl) HDL Cholesterol (mg/dl) Children and Teenagers 4 Age: <10 years old Age: years old Conversion: Conventional units (mg/dl) x = SI Units (mmol/l) Performance characteristics Recovery studies. In two samples with LDL concentrations of 55.2 mg/dl and 41.5 mg/dl different quantities of LDL were added. Subsequent analyses provided recoveries ranging from 98 to 101.7%. The mean proportional systematic error was mg/dl at 130 mg/dl, and mg/dl at 160 mg/dl Method comparison A group of 54 samples were assayed by the proposed method and the ultracentrifugation method. The comparisons yielded a correlation coefficient of and the regression equation was y = 0.96x Total Cholesterol Borderline Adults 3 < Imprecision - Within run Sample 1 Sample 2 Sample 3 N Mean (mg/dl) SD (mg/dl) %CV LDL Cholesterol HDL Cholesterol Optimal Near Optimal Borderline Very Low (desirable) Adults 3 Adults 3 < <40 60 Imprecision - Run-to-run Sample 1 Sample 2 Sample 3 N Analytical sensitivity. Mean (U/L) SD (U/L) %CV Detection limit: mg/dl. The detection limit represents the lowest measurable LDL concentration that can be distinguished from zero. It is calculated as two standard deviations from the mean of 20 replicates of one sample without LDL. Total Cholesterol (mg/dl) Children and Teenagers 4 Age: 2-19 years old Borderline LDL Cholesterol (mg/dl) Children and Teenagers 4 Age: 2-19 years old Borderline < < Notes 1. 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 <0.1mg/L. 03 English - Ref.: 111

4 3. It is suggested to consult to review physiopathological sources and drug interferences in results and methodologies. References 1. Bachorik PS, Ross JW. Clin Chem 1995;41: Westgard JO, Barry PL, Hunt MR, Groth T. Clin Chem 1981; 27: Executive Summary of the Third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of 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. Presentation Application procedures using LDL Liquiform 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 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 - Consumer Service sac@labtest.com.br Product LDL Liquiform Reference 111-1/ Contents 1 X 30 ml 1 X 10 ml 1 X 1,0 ml Revision: July, 2011 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 04 English - Ref.: 111

5 05 English - Ref.: 111

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 Material Tóxico Tóxico Poison 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 Ref.: English - Ref.: 111