血脂分析测试的国家指南

National Guidelines on Lipid Profile Testing E-mail: Dr. Sheng-kai Yan , PhD Department of Laboratory Medicine, Peking Union Medical College Hospital Professor zhou xin Department of Laboratory Medicine, Zhongnan hospital of wuhan university. Preface n Blood lipid analysis has a substantial importance towards the prevention and management of atherosclerosis and coronary heart disease (CHD). n It is also being widely applied to the studies of many other related clinical conditions, such as diabetes mellitus, kidney diseases, and metabolic disorders in postmenopausal women. n Recently, the Lipid Expert Panel of the Chinese Society of Laboratory Medicine (CSLM) of the Chinese Medical Association (CMA) has come up with the practical guidelines and recommendations on lipid profile testing. n lipid profile testing: total cholesterol (TC) triglycerides (TG) high density lipoproteincholesterol (HDL-C) low density lipoprotein-cholesterol (LDL-C) apolipoprotein A1 (ApoA1) apolipoprotein B (ApoB) lipoprotein(a) Lp(a) The Guidelines include the content as follows, n Preface n Preanalytical factors affecting lipid test results n Methods of lipid analysis n Reagent selection criteria and practical guidelines n Clinical significance of cutoff values in the interpretation of lipid profile results See: Chin J Lab Med, 2003, 26(3): 182184 中华检验医学杂志 ,2003,26(3):182-184 Preanalytical Factors Affecting Lipid Test Results n A subjects lipid profile should be measured when the individual is in a steady metabolic state. n Subjects should maintain their usual diet and weight for at least 2 weeks prior to the measurement of their lipids or lipoproteins. n Subjects should not perform vigorous physical activity during the 24 hours prior to testing. Recommendations for minimizing preanalytical variation n Multiple measurements should be performed whthin 2 months, at least one week apart, before making a medical decision about further action. n Fasting or non-fasting specimens can be used for TC testing. However, a 12-h fasting specimen is required for TG and recommended for lipoproteins. n The subject should be seated for at least 5 minutes before specimen collection. n The tourniquet should not be kept on more than one minute during venipuncture. Suggestions to reduce the preanalytical variations on blood lipid profile testing n Both serum and plasma are suitable for analysis. Serum is more common and convenient. The result obtained from EDTA plasma should be corrected for the dilution effect by a factor of 1.03. n The separated serum should be processed as soon as possible. Samples that could not be analyzed within 24 hours, can be stored at 4 for one week or at -20 for several months and at -70 for at least half a year. Repeated freeze and thaw should be avoided. Suggestions to reduce the preanalytical variations on blood lipid profile testing Methods of Lipid Analysis Analytical Methods For routine lipid analysis Serum TC: Enzymatic method(CHOD-PAP method) Serum TG: Enzymatic method( GPO-PAP method) Serum HDL-C: Homogeneous methods Serum LDL-C: Homogeneous methods Serum ApoA1/ApoB and Lp(a): Immunoturbidimetry(ITA) method Immunonephelometry(INA) method (The first choice would be ITA, followed by INA) Serum HDL-C Homogeneous methods n Clearance method Synthetic polymer/ detergent HDL-C assay, SPD Daiichi Pure Chemicals Co. Catalase HDL-C assay, CAT Denka Seiken Randox Co. Reference Diagnostics Polymedco Serum HDL-C Homogeneous methods n PEG-modified enzyme HDL-C assay， PEGME Kyowa Medex Co. Roche Diagnostics Centronic GmbH n Immunoseparation method, IS International Reagents Corp HDL-C assay， IRC International Reagents Co./Sysmex Antibody immunoseparation HDL-C assay ， AB Wako Pure Chemicals Industry Serum HDL-C Homogeneous methods n Polyanion Polymer/ detergent HDL-C assay ， PPD Daiichi Pure Chemicals Co. Genzyme Diagnostics Serum HDL-C Homogeneous methods n Clearance method Surfactant LDL-C assay， SUR Daiichi Pure Chemicals Co. Genzyme Diagnostics Catalase LDL-C assay， CAT Denka Seiken RANDOX Polymedco Kyowa Medex Roche Serum LDL-C Homogeneous method n Protecting reagent LDL-C assay， PRO Wako Chemicals Sigma Diagnostics n Calixarene LDL-C assay， CAL International Reagents Co./Sysmex n Solubilization LDL-C assay， SOL Serum LDL-C Homogeneous method Serum Lp(a) Immunoturbidimetry/immunonephelometry The reagent should preferably be polyclonal or mixed monoclonal antibodies, that could recognize different epitopes of the Apo(a) molecule. ITA is more preferable than INA. n Spectrophotometers and semi-/automatic biochemical analyzers would be suitable for analysis once verified for proper functioning. All samplers, dilutors, pipettes and micropipettes must be calibrated. n Automatic biochemical analyzers (fully or semi-automatic) are recommended for use in blood lipid testing. Requirements on Analytical Instruments n Parameters should be set according to the manufacturers instructions and assigned calibrator values on the package insert. n The parameters should not be liberally changed. Setting the Parameters n Laboratories should use accredited quality control (QC) materials with serum matrix as the internal quality control. n The QC materials should cover both the normal and pathological ranges. n There are QC materials specifically designed for lipid analysis that laboratories could consider to purchase. Quality Control n While choosing individual QC materials, one should consider carefully the dynamic range and the target values for the corresponding analytical methods. n A parallel run should be performed for an overlapping period with both the current and new lot control materials. n Enrolment to external quality assessment programs is a MUST. Quality Control Reagent Selection Criteria and Practical Guidelines Inaccuracy and Imprecision Inaccuracy(Bias) Imprecision(CV) Total errors * TC 3% 3% 8.9% TG 5% 5% 15% HDL-C 5% 4% 13% LDL-C 4% 4% 12% ApoAI 3% 5% ApoB 3% 5% Lp(a) 4% 10% *Total errors=Bias%+1.96CV Analytical Sensitivity n When phenol is employed in enzymatic analysis of serum TC, the absorbance of TC = 5.2 mmol/L at 500nm (A500nm) is about 0.30-0.35. Therefore, A500nm of 0.005 should give 0.08 mmol/L TC. n The sensitivity of TG enzymatic analysis should be A500nm 0.2 at TG 2 mmol/L. Analytical Sensitivity n In using homogeneous assays for HDL-C and LDL-C, the minimal measurable level should be 0.01 mmol/L. n The lowest detection limits for serum ApoA1 and ApoB by immunoturbidimetry or immunonephelometry should be 0.5 g/L, and that for Lp(a) should be 5 mg/L. Linearity n The upper limit of linearity is 13 mmol/L when using the dilution ratio of 1:100 in enzymatic analysis of TC. It will lower the upper limit if smaller dilution ratios are used. n The linearity of the enzymatic TG assay should at least be 11.3 mmol/L (1000 mg/dL). n The linearity of homogeneous assays for HDL-C and LDL-C should at least be 2.59 mmol/L and 7.77 mmol/L respectively. Linearity n The linearity of serum ApoA1 and ApoB by ITA or INA should not be less than 2.0 g/L and that of Lp(a) not less than 800 mg/L, respectively. Specificity n In enzymatic analysis of serum TC, the color reaction is subject to certain degree of spectral interferences from various non-cholesterol sterols. Normally, there is only negligible amount (about 1%) of these non-cholesterol sterols in the blood. n In enzymatic analysis of TG, the lipoprotein lipase (LPL) can hydrolyze TG and also mono- glycerides and di-glycerides. The latter two constitute about 3% of total TG measured. Specificity n The recoveries in homogeneous assays of HDL-C and LDL-C, immunoturbidimetry of serum ApoAI, Apo B and Lp(a) should preferable be in the range of 90%-110%. In general, the measurements should not be affected by other lipoproteins. n There is no significant interference up to hemoglobin concentration of 2g/L, bilirubin concentration of 0.1g/L in enzymatic analysis of serum TC. n Interference in enzymatic analysis of TG is similar to that of the TC assay. There will be negative interference when bilirubin 100mol/L or ascorbic acid170mol/L. Hemoglobin will cause spectral interferences. Grossly hemolysed samples are not suitable for analysis. Interferences n There is no significant interference of TG 5.65 mmol/L (500 mg/dl), bilirubin 513 mol/L (30 mg/dl), and Hb 5 g/L in most homogeneous assays for HDL-C and LDL-C, as well as the immunoturbidimetric or immunonephelomentric assays for serum ApoA1, ApoB and Lp(a). Interferences Reagent Stability n Lyophilized reagents can usually be stored at least for 1 year if kept unopened at 2 8 . Reconstituted cholesterol and TG enzymatic reagents should be stored at 2 8 for 2 days. n Reagents should be discarded if pink color is seen. The absorbance at 500nm of the reagent blank should be 0.05. n Unopened reagent solutions should be stable for at least 6 months at 2 8 and for 1 month after being opened. Reaction Rates n The reaction should not be longer than 5 min and 8 min at 37 for enzymatic analysis of serum cholesterol and TG respectively. n The endpoint of immunoturbidimetric assays for serum ApoA1, ApoB and Lp(a) could be determined according to their reaction curves shown in the automatic analyzer. Generally, reaction time of 8 10 minutes is acceptable. Calibration n The user should use the calibration materials provided by the manufacturer. The calibration materials should be traceable to the reference methods. n One should avoid using different brands of calibration materials as to ensure consistence of the performance. Calibration n The international standard serum