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From The British Journal of Diabetes and Vascular Disease

When and How to Measure Lipids and Their Role in CHD Risk Prediction
Posted 09/22/2003 William Richmond

Abstract and Introduction

Abstract Accurate lipid and lipoprotein measurements, control of pre-analytical factors and consideration of biological variability are required for the reliable assessment of coronary heart disease (CHD) risk and the management of dyslipidaemia. Inclusion of high density lipoprotein-cholesterol, in addition to total cholesterol, in risk calculations is preferred to attain good sensitivity and specificity. Although the strong and independent association of triglyceride with CHD has been confirmed, its large biological variation limits its utility as a risk factor or primary treatment target. A fasting profile including triglyceride is essential however for consideration of genetic and secondary causes of dyslipidaemia and decisions on therapeutic intervention. Introduction Lipid measurements are integral components of risk prediction in the primary prevention of cardiovascular disease and management of therapy in the primary and secondary prevention of coronary heart disease (CHD). This article reviews the application of lipid measurements in the current evidence-based guidelines issued for this purpose. It also discusses the required quality of the laboratory data and its appropriate application in risk assessment and therapeutic intervention

Lipid and Lipoprotein Measurements in Risk Assessment

Total and Lipoprotein Cholesterol Raised total cholesterol, low density lipoprotein-cholesterol (LDL-c) and reduced high density lipoprotein-cholesterol (HDL-c) levels have all been shown to be strong independent risk factors for CHD in numerous cross-sectional, prospective and intervention trials. In multivariate analysis of the Framingham study data, HDL-c has been shown to be the best single lipid/lipoprotein predictor of cardiovascular and CHD mortality [1] while LDL-c predicts CHD only marginally better than total cholesterol. Triglyceride On univariate analysis triglyceride has been shown to be a strong risk factor for CHD. There is conflicting evidence, however, on the independence of this association on multivariate analysis. [2] In some studies triglyceride was an independent CHD risk factor only when HDL-c was low [3] or when the LDL-c:HDL-c ratio was high, largely due to a low HDL-c. [4,5] It has been demonstrated, however, that the greater biological variation of triglyceride (21%), which is three-fold that for HDL-c, is such that a single measurement is not representative of the mean value in an individual and consequently erodes the relationship between triglyceride and CHD incidence on multiple

logistic regression analysis.[6,7] When this is corrected for, by using true mean values for individuals, the strength and independence of triglyceride as a risk factor is confirmed. [8] Its utility in risk calculations however remains limited by its biological variability and the requirement for a fasting value. The Atherogenic Lipoprotein Phenotype Although triglyceride-rich lipoproteins may have a direct influence on atherogenesis in the arterial wall, their role as metabolic determinants of reduced HDL-c concentrations and the production of atherogenic small dense LDL is of considerable significance. This effect can occur at very modest elevations of triglyceride and low-risk levels of total and LDL-c. The resulting atherogenic phenotype, characterised by mild hypertriglyceridaemia, low HDL-c and an increased preponderance of small dense LDL, frequently occurs in insulin-resistant states and is reported to be the dyslipidaemia most commonly associated with CHD.[9] The inclusion of HDL-c in risk assessment calculations is therefore an important and effective means of incorporating the atherogenic influence of triglyceride through its strong and inverse relationship with HDL-c. Unlike triglyceride this measurement can be made in the non-fasting state and, as indicated above, has a smaller biological variation. LDL Composition Small dense LDLs are cholesterol depleted, consequently measurements of total- or LDL-c do not accurately reflect the numbers of these atherogenic particles. Each lipoprotein particle in the very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and LDL density range carries one molecule of the apolipoprotein (apo) B. In most circumstances > 90% of plasma apo B is associated with the LDL fraction. Apo B concentrations are consequently an excellent index of LDL particle number and have therefore been proposed as a superior marker of CHD risk than total or LDL-c. [10,11] There are however no prospective data available from studies such as Framingham to enable apo B to be included in global risk calculations. Total Cholesterol:HDL-c Ratio Of all the risk factor variables the total cholesterol:HDL-c ratio has been shown to be the best predictor of CHD at any total cholesterol level[1,12] and second only to the Framingham logistic model in its predictive power.[13] This is not surprising in view of the strong independent and opposing influences of these variables and is strong evidence for the desirability of using the total cholesterol:HDL-c ratio in risk prediction calculations.

Lipid and Lipoprotein Measurements in Current Guidelines

A number of guidelines have been published following the demonstration of the safety and efficacy of cholesterol lowering using statins in primary and secondary prevention trials. These guidelines include point score tables or charts to quantify absolute risk of CHD or cardiovascular disease (CVD) risk in primary prevention and direct therapeutic intervention. There are currently noticeable differences between these guidelines in: the lipid and non-lipid risk factors included in the risk calculation (table 1)

the risk threshold at which intervention is recommended the treatment targets for different levels of risk (table 2).

The International Task Force for prevention of CHD/International Atherosclerosis Society (ITF/IAS)[14] These guidelines use point scoring tables derived from a Cox proportional Hazards model calculated from the 10-year follow-up data of a cohort of 5,389 men (35-65 years of age) in the PROCAM study. Increased risk is graded as small, moderate or high when point scores fall into the 3rd, 4th or 5th quintiles of the 10-year CHD incidence distribution. The PROCAM risk calculator is also available as an interactive program on the Task Force website (www.chd-taskforce.com). This scoring system is the only one to include fasting triglyceride, LDL-c and family history of premature myocardial infarction. It only applies to men, however. The US National Education Program (NCEP) Adult Treatment Panel III (ATP III) [15] These most recent guidelines include total cholesterol and HDL-c in point score tables derived from a stepwise logistic regression function applied to the Framingham 12-year follow-up data of 12,000 men and women. Diabetes is classified as a CHD equivalent in these guidelines and is therefore not included in the risk assessment for primary intervention. Although the risk assessment can be conducted on non-fasting individuals these guidelines recommend an initial fasting investigation, if possible. This enables triglyceride and LDL-c assessment to be made at the outset and assists in the early characterisation of any dyslipidaemia identified. The full Framingham function is also available as a computer program. In assigning categories for therapeutic intervention the number of risk factors present is taken into account in addition to the 10-year risk score (table 1). In addition to its inclusion in the risk score a low HDL-c (< 1.04 mmol/L, 40 mg/dL) is counted as a risk factor while a high HDL-c (>/= 1.6 mmol/L, 60 mg/dL) is classed as a negative risk factor and subtracted from the total. The Joint European Societies (JES),[16] Joint British Societies (JBS)[17] and New Zealand (NZ) guidelines[18] These guidelines all include risk assessment charts derived from the Framingham multiple logistic function. The JES and JBS charts provide 10-year CHD risk predictions. The NZ charts calculate five-year CVD risk with the inclusion of systolic and diastolic blood pressure (BP). Use of 2-dimensional charts in the JES risk assessment enables total cholesterol and systolic BP to be used as continuous quantitative variables as opposed to categorical variables when point scores tables are used. This advantage is exploited further in the JBS and NZ charts by using total cholesterol and HDL-c as a ratio thereby incorporating three continuous variables (systolic BP, total cholesterol and HDL-c) in 2-dimensional charts which are very easy to use. The JBS cardiac risk calculator is also available as a computer program on the British Hypertension Society website (www.hyp.ac.uk/bhs).

The JES guidelines do not include HDL-c as a variable in the risk calculation but substitute a fixed value (1.0 mmol/L for males; 1.1 mmol/L for females) in the calculation. Exclusion of HDL-c in this manner has been shown to markedly reduce the sensitivity[19] and accuracy[20] of risk assessment risk calculations. Although the JES, JBS and NZ Framingham based risk calculations do not include triglyceride, these guidelines do acknowledge the increased risk associated with an elevated triglyceride (> 2.3 mmol/L, 200 mg/dL), particularly when it co-exists with a low HDL-c (< 1.0 mmol/L) or elevated total cholesterol:HDL-c ratio (> 5.0). It is recommended that, although a non-fasting sample is adequate for initial assessment, a fasting follow-up lipid profile including triglyceride is indicated if other risk factors are identified or any abnormality in total- or HDL-c is noted in the initial investigation. Comparison of Risk Prediction Calculation Results The Framingham equation and PROCAM function give broadly similar results. However, in comparison with the Framingham equation, the PROCAM function has been shown to underestimate risk at lower levels but overestimate risk at higher levels in a study involving UK male patients with and without diabetes.[21] Limits on age and the measured lipid and non-lipid variables also excluded a large number of patients for assessment by the PROCAM equation. A comparison of all the published Framingham-based risk tables and charts with the full Framingham equation as the reference method demonstrated that the JBS charts appear to have the best combination of sensitivity, specificity and ease of use in primary care patients. [22] It is suggested however that the available computer programs allowing direct calculation with the Framingham equation have the advantage of greater accuracy. In addition they allow left ventricular hypertrophy to be taken into account and provide both coronary and stroke risk based on systolic and diastolic BPs.[23]

Identification of Familial Dyslipidaemia and Identification of Secondary Causes

Implicit in all screening and risk assessment procedures is that when dyslipidaemia is identified consideration should be given to genetic and secondary causes with family screening and referral to specialists when appropriate. Identification of familial hypercholesterolaemia (FH) is particularly important in view of the autosomal dominant inheritance of this disorder and its high prevalence. FH affects one in 500 of the population in its heterozygous form and untreated 50% of male and 15% female heterozygotes die of CHD before age 60. This risk is underestimated by the Framingham equation because these individuals frequently do not have other risk factors and have been exposed to high cholesterol concentrations since childhood. Family screening of affected probands is therefore the most efficient way of detecting individuals in this high-risk group.[24]

Intervention Thresholds, Lipid Treatment Targets and Therapeutic Goals

Although all guidelines agree that those with established CHD or familial dyslipidaemia should be treated without risk estimation, only the ATP III guidelines classify diabetes as a CHD equivalent in this respect. Intervention Thresholds

The level of risk at which drug intervention is recommended, when lifestyle modification proves inadequate, clearly has significant resource implications for healthcare providers. For primary prevention, the ATP III intervention threshold for statin therapy, which is dependent on LDL-c levels in addition to a defined risk category, is equivalent to a 10year CHD risk of >/= 10%. The JES risk threshold is >/= 20%. The JBS recommended threshold is intermediate at >/= 15% but, in recognition of the resource implications for the National Health Service (NHS), a progression from an initial risk threshold of >/= 30% to >/= 15% over a 10-year period was suggested.

The UK National Service Framework on CHD prevention however recommends the use of statins in primary prevention only when the 10-year CHD risk is >/= 30% and has outlined a prevention strategy identifying priority groups for lipid screening, global risk assessment and therapeutic intervention.[25] Those under 75 years of age with established coronary, cerebro- or peripheral-vascular disease or multiple risk factors are first priority. Second priority includes individuals with hypertension, diabetes, family history of premature CHD, first degree relatives of probands with FH and other familial dyslipidaemias with significant risk. Finally, as resources allow, it is intended to extend screening for lipid and other risk factors to all adults. Treatment Targets and Therapeutic Goals The primary lipid targets and therapeutic goals for the risk categories defined in the ITF/IAS, ATPIII, JBS and JES guidelines are summarised in table 2. The primary treatment target in the ITF/IAS and ATP III recommendations is LDL-c and the therapeutic goals are graded according to the degree of risk assigned. Particular emphasis is placed on achieving low LDL levels (< 2.6 mmol/L,100 mg/dL) in secondary prevention and highrisk individuals in primary prevention. The JBS and JES recommendations are much simpler in applying the same treatment goals (total cholesterol < 5.0 mmol/L and LDL-c < 3.0 mmol/L), for secondary prevention and all levels of risk in primary prevention. They are less stringent, however, than the ITF/IAS and ATP III guidelines in secondary prevention and high-risk individuals. The ATP III guidelines, in recognition of the emergence of triglyceride as an important influence on CHD risk, identify non-HDL-c (total cholesterol minus HDL-c) as a secondary treatment target in individuals whose LDL treatment goal has been achieved but whose triglyceride remains elevated (> 2.3 mmol/L). Non-HDL-c is a measure of cholesterol in VLDL and their atherogenic lipolytic products in the LDL fraction. The non-HDL-c goals are given as the LDL-c goal for a particular risk category + 0.8 mmol/L (30 mg/dL) - an estimate of VLDL cholesterol by the Friedwald formula when triglycerides are normal (1.7 mmol/L). In ATP III, triglyceride becomes a primary treatment target in patients with very high fasting triglyceride (> 5.6 mmol/L, 500 mg/dL) to prevent pancreatitis. Consideration is also given to HDL elevation in CHD or CHD equivalent patients whose triglyceride is normal (< 1.7 mmol/L, 150 mg/dL) and whose LDL-c and Non-HDL-c treatment goals have been achieved but whose HDL-c remains low (< 1.04 mmol/L, 40 mg/dL).

Required Analytical Quality of Lipid Measurements

Maximum Allowable Total Error The total error (TE) in an analytical procedure combines the influence of bias and imprecision (CVanalytical) and can be calculated, within 95% confidence limits, from the formula: TE = Bias(%) + 1.96 CVanalytical The NCEP have made recommendations for the maximum allowable TE%, within 95% confidence limits, to permit the effective clinical application of lipid measurements in risk assessment and therapeutic intervention (table 3). To achieve this analytical goal for TE, laboratories can calculate the maximum bias allowable for the imprecision achieved by their analytical systems and ensure this is attained by appropriate calibration and quality assurance measures. Accuracy When analyte concentrations are used to calculate risk, set treatment thresholds and assign therapeutic goals, it is evident that laboratories providing measurements for this purpose must use the same accuracy base upon which the epidemiological and clinical trial evidence for these decision limits was obtained. The Framingham lipid data and other studies from which the medical decision points recommended by ATP III and other advisory committees, worldwide, are based on measurements standardised by the Centers for Disease Control and PreventionNational Heart, Lung and Blood Institute (CDC-NHLBI) Lipid Standardisation Program (LSP). [26] This accuracy base has won universal acceptance for total cholesterol, HDL-c, LDL-c and triglycerides. Consequently lipid measurements provided for CHD risk assessment and therapeutic intervention are required to have proven accuracy on patient samples which is traceable to the CDC reference methods. The complexity of lipoproteins and their heterogeneity in plasma presents a considerable challenge in the development of definitive and reference methods and the reliable transfer of accuracy from these to routine methods using reference materials. The lipoproteins and their lipid components in these materials can exhibit different reactivity to those in native serum samples in routine assays. These matrix effects have necessitated the establishment of a network of CDC approved reference laboratories[27] which provide a service to diagnostics manufacturers to validate the calibration of their lipid and lipoprotein assays. This is achieved by comparison of results between reference and routine methods on fresh serum samples and assigning values to calibration materials used in these systems which will ensure their accuracy. Laboratories should be aware of the potential of matrix effects in calibration and quality control materials and adhere to the use of certified systems whose accuracy is traceable to the accepted CDC accuracy base in this manner. Quality Assurance Schemes are also required to ensure that matrix effects are minimised in their materials. Analytical Imprecision and Biological Variation Recently reported estimates of within individual biological variation (CV biological) for key lipids and lipoproteins[28] are listed in table 4 together with the NCEP recommended goals for precision for these analytes. From these figures the contribution of analytical imprecision to the total result variability has been calculated from the formula given by Fraser and Harris [29] and is shown to range from < 3% for triglyceride, through < 12% for total cholesterol and LDL-c, to < 15% for HDL. This is in line with the recommendation of Harris that it is desirable that CV analytical should be less than 50% of CVbiological on the empirical basis that total result variability due to the analytical

component is less than 11.8% in such circumstances.[29] The clinical utility of an investigation is then largely determined by the individual biological variation of the parameter. On the basis of these figures for biological variation and acceptable imprecision, the Smallest Significant Difference (SSD%) between consecutive measurements has been calculated, together with the number of serial results which must be averaged to estimate the Homeostatic Set Point (HSP) of an individual to a given degree of accuracy. [29] (table 4). These statistics are an important consideration in use of lipid and lipoprotein measurements to assess risk, response to therapy or attainment of a therapeutic goal: The SSD between consecutive measurements (p<0.05) is approximately 15% for total cholesterol, 20% for HDL-c and LDL-c and 50% for triglycerides. A single measurement will be within 15% of the HSP for total cholesterol, within 20% for HDL-c and LDL-c. To achieve a more accurate estimate within 10%, however, requires the mean of two, three and four measurements respectively for these analytes. For triglyceride, which has a much greater biological variation, the mean of two results will estimate the HSP to within 30% and it requires five results to be within 20%.

Lipid Measurement: Biological Variation

Minimising Sources of Pre-Analytical Variation Careful attention to patient preparation and blood collection technique can do much to facilitate accurate assessment of patients by controlling factors which induce variability in lipid and lipoprotein concentrations. These include: Prandial status: A 12-hour fast essential for triglyceride measurement and calculated LDL, but not essential for total- or HDL-c measurements Lifestyle: The patient should be metabolically stable and advised to maintain their normal diet, alcohol intake, smoking and exercise habits prior to testing Pregnancy or illness: Investigation should be deferred for two weeks after a minor illness, 2-3 months after pregnancy, major illness or myocardial infarction. A sample taken within 24 hours of the onset of myocardial infarction, however may match the pre-infarction state Phlebotomy: Increases in lipid concentrations of up to 15%, resulting from diffusion of water from blood vessels into tissues, can occur within 15 minutes of changing from a supine to a standing position or if a tournique is applied for more than five minutes. Blood samples should therefore be drawn after the patient has been seated for at least five, preferably 15 minutes. If necessary, a tournique should not be applied for more than one minute Multiple Measurements: As discussed earlier, medical decisions should be based on the required number of measurements performed within two months and at least one week apart. To avoid interlaboratory differences in bias, these measurements should be performed by the same laboratory

Analytical Methods
Total Cholesterol Approximately 75% of cholesterol transported in the plasma lipoproteins is esterified to fatty acids. All procedures involve the hydrolysis of cholesterol ester and measurement of the total free cholesterol. The CDC accuracy base for cholesterol comprises a primary reference (definitive) isotope dilution mass spectrometry (IDMS) procedure, a certified pure cholesterol standard and a secondary reference method involving chemical hydrolysis, solvent extraction and colour reaction. Routine enzymatic reagent systems for total cholesterol measurement contain detergents and lipases which disrupt lipoprotein complexes and hydrolyse cholesterol esters. Enzymic oxidation of the liberated cholesterol produces equimolar amounts of hydrogen peroxide which is measured in a peroxidase catalysed colour reaction. Triglyceride In addition to lipoprotein triglyceride, plasma contains small amounts of mono- and di-glycerides and free glycerol. As usually applied routine methods hydrolyse all these glycerides to release glycerol and measure total glycerol. Although there is some concern about the inclusion of endogenous glycerol in the measurement, this is not a problem in out-patients when levels are usually < 0.1 mmol/L.[30] Levels can be higher in in-patients, however, particularly in those receiving glycerol containing lipid emulsions for parenteral nutrition, following heparin infusions, in diabetic ketoacidosis and in liver disease.[30] The CDC triglyceride accuracy base comprises primary reference procedure, a certified triglyceride standard and two secondary reference methods. The IDMS primary reference method measures total glycerides and free glycerol. One secondary reference is an IDMS procedure which measures only free glycerol. The other, a chemical procedure, involving hydrolysis, extraction, purification and a colour reaction, measures only mono-, di- and triglycerides. Taken together, the reference procedures enable free glycerol concentration to be monitored in calibration and control materials while assigning total glyceride values. In routine enzymic procedures, in a very similar sequence of reactions to that for the measurement of cholesterol, triglyceride is released from lipoproteins and enzymically hydrolysed to glycerol and fatty acids. The glycerol released is then phosphorylated and measured in an enzyme linked colour reaction. It is possible to exclude glycerol from the measurement using blanking techniques. This involves additional cost and is not employed in the majority of routine laboratories. Lipoprotein Measurements The heterogeneous nature of lipoprotein density classes precludes the preparation of certified standards and the development of definitive methods. Consequently the accuracy base for HDLc and LDL-c is dependent on the assignment of reference values to serum pools by a designated secondary reference method. HDL cholesterol. HDL is defined as the lipoprotein particles obtained by preparative ultracentrifugation in the density range 1.006-1.21 kg/L. The CDC reference procedure for HDL-c is a three-step process in which VLDL is removed by ultracentrifugation, LDL is selectively

precipitated from the remaining sample and HDL-c is measured in the remaining supernatant using the CDC reference cholesterol method. Due to the large sample volume required for this procedure and its complexity, a simpler selective precipitation method has been standardised by the CDC reference procedure and validated for use as a designated comparison method by reference laboratories. In this method VLDL and LDL are precipitated together using 50-Kda dextran sulphate and magnesium ions. HDL-c is then measured in the supernatant using the CDC reference cholesterol method. Although several selective precipitation procedures have been used, a major advance in the routine measurement of HDL-c has been the development of homogeneous reagent systems in which HDL-c can be measured without prior isolation of HDL from other lipoprotein classes. This technology employs various strategies involving differential detergent, surfactant or antibody interactions with the plasma lipoproteins to allow the enzymatic components of cholesterol reagent, described above, to react selectively with HDL particles. These homogeneous assays have been shown to be well suited for routine use in clinical laboratories, generally meeting the NCEP criteria for precision, accuracy and total error. However, discrepant results have been reported with homogeneous HDL assays in samples with atypical lipoprotein characteristics e.g. type 3 hyperlipidaemia. LDL cholesterol. The traditional density classification of LDL is 1.019-1.063 kg/L and includes Lp(a). The procedures used to establish the LDL cholesterol decision limits used in all current guidelines, also include intermediate density lipoproteins (IDL) 1.006-1.019 kg/L in the measurement. Density Lipoprotein < 1.006 kg/L 1.006-1.019 1.019-1.063 1.063-1.21 VLDL IDL + LDL revised LDL definition LDL-c, according to this revised definition, can therefore be determined as: LDL-c = Total cholesterol - (HDL-c) - (VLDL-c). This principle is applied in the CDC reference method and recommended routine method as follows: In the CDC reference procedure for LDL-c, 'beta quantitation', cholesterol is measured in a sample after removal of VLDL by ultracentrifugation and again after selective precipitation of LDL. The first cholesterol measurement therefore represents LDL + HDL-c and the second measurement HDL-c. LDL-c is therefore determined by the difference between the two values. Friedewald Equation. The routine procedure currently recommended by the CDC uses the formula developed by Friedewald in which VLDL cholesterol is estimated as plasma triglyceride/2.19 in mmol/L units or 5.0 in mg/dL. Thus LDL-c can be calculated using routine laboratory measurements of total cholesterol, HDL-c and triglyceride: HDL

LDL-c = Total cholesterol - (HDL-c) - (Triglyceride / 2.19). This estimate is not valid when chylomicrons are present (non-fasting), when triglyceride levels are > 4.52 mmol/L (400 mg/dL) or in individuals with type 3 hyperlipidaemia. In these circumstances VLDL cholesterol is overestimated and LDL consequently underestimated. This estimate is not valid when chylomicrons are present (non-fasting), when triglyceride levels are > 4.52 mmol/L (400 mg/dL) or in individuals with type 3 hyperlipidaemia. In these circumstances VLDL cholesterol is overestimated and LDL consequently underestimated. Concordance with the reference method is best when serum triglycerides are < 2.3 mmol/L and acceptable up to 4.52 mmol/L.[31] There has been some concern that this calculation is not valid in diabetic individuals. It has been demonstrated, however, that the apparent greater discrepancy between the Friedewald and reference values in diabetic compared with non-diabetic patients is a reflection of the increased proportion of diabetic patients with triglycerides in the 2.34.52 mmol/L range.[31] It has been reported that the Friedewald formula underestimates LDL-c at low concentrations.[32] This may be a consideration in monitoring the attainment of therapeutic goals on statin treatment and there is some evidence that outcomes are more accurately reflected by apo B measurements in this situation. [33] A further concern is that the calculation of LDL-c involves the imprecision and bias inherent in three measurements. The contribution of HDL to total error is reduced due to its lower magnitude in relation to total cholesterol. Similarly, the impact of the large biological variation of triglyceride will be diminished by the divisor in the Friedewald estimate of VLDL cholesterol. Nonetheless it is necessary for laboratories to be within the NCEP analytical goals for each of the three variables to achieve that for calculated LDL-c.

Future Methods. The shortcomings of LDL-c calculation have provided impetus to the development of homogeneous LDL-c assays. These have proved to be analytically more challenging and complex than the homogeneous HDL assays. While they show great promise, and would enable LDL-c to be measured in the non-fasting state, it has been recommended that they require more validation in a range of dyslipidaemias before they can be fully endorsed for routine use.[31] While automated apo B methods are available and an accepted standard has been defined by the International Federation of Clinical Chemistry (IFCC), there is as yet no accepted reference method to serve as an accuracy base and the matrix sensitivity of the systems and their reliability in different lipoprotein phenotypes remains to be confirmed.

The clinical utility of lipid and lipoprotein measurements is determined by their biological variation, the control of pre-analytical factors influencing variability and the precision and accuracy with

which they can be measured (table 5). The mean of three estimations is required to accurately assess total-cholesterol, HDL-c and LDL-c, while several estimations are required for triglyceride. The large biological variation of triglyceride erodes its value in risk calculations and introduces variability in the calculation of LDL-c. This together with the requirement for fasting measurements has led to the more general use of HDL-c and total cholesterol for this purpose and the total cholesterol:HDL-c ratio has been identified as a particularly strong predictor of CHD risk. The inclusion of HDL-c has been shown to be essential for the achievement of specificity and sensitivity in risk calculations and the direct use of risk equations in computer programs, more accurate than charts or point score tables. While LDL-c remains the primary target for therapy, the atherogenic influence of triglyceride is increasingly recognised. Risk assessment strategies and thresholds for drug intervention are strongly influenced by their resource implications in healthcare provision. Reprint Address Correspondence to: Dr William Richmond Chemical Pathology, St Mary's Hospital, Praed Street, Paddington, London, W2 1NY, UK. Tel: +44 (0)207 886 1374; Fax: +44 (0)207 886 1904. E-mail: w.richmond@imperial.ac.uk Abbreviation Notes Apo = apolipoprotein; BMI = body mass index; BP = blood pressure; CHD = coronary heart disease; CVD = cardiovascular disease; FH = familial hypercholesterolaemia; HDL-c = high density lipoprotein-cholesterol; HSP = homeostatic set point; IDL = intermediate density lipoprotein; IDMS = isotope dilution mass spectrometry; LDL-c = low density lipoproteincholesterol; NHS = National Health Service; SSD = smallest significant difference; TE = total error; VLDL-c = very low density lipoprotein-cholesterol Trial Acronyms PROCAM = PROspective CArdiovascular Mnster study