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Advances in the measurement of LDL cholesterol

1st April 2013


Until recently, direct measurement of LDL cholesterol was not practically possible for most laboratories in a routine clinical setting. The first homogeneous LDL method was introduced in 1997. Here, Mauro Panteghini looks at the clinical significance and advances in measurement of LDL cholesterol.

Increased low density lipoprotein (LDL) cholesterol constitutes a major risk factor for the development of coronary heart disease. The relationship between LDL cholesterol and coronary artery disease has been established in many observations and epidemiological studies, together with numerous clinical trials of lipid lowering intervention studies, including diet, drugs and partial ileal by-pass surgery.

These clinical trials of lipid lowering therapy have demonstrated conclusively that decreasing LDL cholesterol decreases coronary artery disease morbidity and mortality, slows disease progression and can alleviate the progression of artherosclerotic lesions.

The first homogeneous LDL method was introduced by Genzyme Diagnostics at the end of 1997. This assay measures LDL cholesterol without the need for any off-line sample pre-treatment or centrifugation. It requires only three microlitres of sample and comprises two ready to use liquid reagents. In the first part, lipoproteins other than LDL are disrupted by a specific detergent and the released cholesterol is consumed by cholesterol enzymes in a colourless reaction.

Subsequently, a second detergent destabilises the remaining LDL particles and the chromogenic coupler produces a colour which is proportional to the amount of LDL cholesterol.

Prevention trials

The first trial to show a reduction in coronary events with LDL cholesterol reduction was the Lipid Research Clinics Coronary Primary Prevention Trial published in 1984 in which about 3800 men with LDL cholesterol higher than 190 mg/dL were randomly selected to receive the resin cholestyramine or placebo. Total cholesterol was decreased by 8 per cent and LDL by 20 per cent from base line with cholestyramine. The combined primary end point of definite cardiac death and of definite non-fatal myocardial infarction was significantly decreased by 19 per cent.

The advent of hydroxy methyl glutaryl coenzyme A reductase inhibitors, the so-called statins, made possible greater LDL cholesterol reductions by about 20 to 45 per cent because of increased efficacy and improved acceptability to the patient. Statins inhibit HMG Co A reductase, the rate limiting enzyme of cholesterol biosynthesis. The compensatory increase in hepatic LDL receptor activity increases clearance of LDL particles and results in decrease of LDL concentration in plasma.

The effects of statin therapy on coronary morbidity and mortality have been recently examined in clinical trial in which the primary end point was clinical events.

Five landmark studies have proven that lowering the LDL cholesterol in patients with heart disease but also in those without prior evident coronary disease can reduce major cardiac events by as much as 30 per cent.

Treatment of an elevated LDL cholesterol in those without prior coronary disease is called primary prevention, while treatment in those with prior coronary disease is secondary prevention.

Primary prevention of coronary events was studied in patients with high LDL cholesterol in the West of Scotland Coronary Prevention Study (WOSCOPS) and in patients with mild to moderate LDL elevation in the Air-Force (Texas) Coronary Atherosclerosis Prevention Study (AFCAPS). Secondary prevention was studied in patients with high LDL cholesterol in the Scandinavian Simvastatin Survival Study (4S) and in patients with mildly to moderately elevated LDL cholesterol in the Cholesterol and Recurrent Events (CARE) trial and in Long Term Intervention with Pravastatin in the Ischemic Disease (LIPID) study.

These studies which enrolled a total of more than 30 000 patients consistently demonstrated a benefit on relative risk reduction. Based on this accumulation of evidence, LDL cholesterol is the primary focus for the diagnosis and treatment of hypercholesterolemia in the adult treatment guidelines of the US National Cholesterol Education Program (NCEP). The NCEP guidelines stratify patients based on risk for a coronary event with progressively lower LDL cholesterol concentrations recommended for patients at increasingly higher risk.

The NCEP guidelines recommend the LDL cholesterol concentration be lower than 100mg/dL in patients with known coronary disease. Also at high risk are primary prevention patients with multiple risk factors. The target of therapy in these patients is to lower LDL cholesterol to less than 130mg/dL.

Accordingly, physicians should use LDL cholesterol concentrations to determine if the patient had a lipid abnormality and whether dietary or drug therapy should be instituted. They should also identify the treatment goal, how close the patient is to this goal and whether the patient is responding to treatment. Because the LDL cholesterol concentration is the basis for the patient's assessment and treatment algorithm, simple and automated methods should be widely available to directly measure LDL cholesterol in serum.

Until recently, direct measurement of LDL cholesterol was not practically possible for most laboratories in a routine clinical setting. The beta quantification procedure based on ultra-centrifugation is still the accepted reference method. It is time consuming, labour intensive and unsuitable for use in a clinical laboratory. Furthermore it is an indirect measure of LDL cholesterol.

A direct method for measuring LDL cholesterol with an immunoseparation reagent was developed some years ago.

The method uses latex beads coated with polyclonal anti-serum to specific apolipoproteins to remove HDL and VLDL from samples.

After centrifugation the LDL cholesterol remaining in the filtrate was quantified by an enzymatic cholesterol assay. This method required time consuming centrifugation and expensive anti-serum.

In the absence of simple methods for directly measuring LDL cholesterol the Friedwald calculation method has been widely employed to provide an estimate of the LDL cholesterol values. However, the calculation suffers from a number of well known drawbacks. An error in one test component adversely affects the overall result. Accuracy drops dramatically when triglyceride is above 200mg/dL. The patient must fast before testing to avoid triglyceride bias. LDL cholesterol may be grossly over-estimated or under-estimated in patients with different types of hyperlipidemia.

In 1990, a study by Tufts University in Boston compared calculated LDL cholesterol values with measurements by ultracentrifugation in about 5000 fasting adults.

The formula misclassified about 15 per cent of patients with triglyceride concentrations lower than 200mg/dL, 23 per cent of patients with triglyceride between 200 and 300mg/dL and 41 per cent of patients with triglyceride between 300 and 400mg/dL.

In recognition of these problems four years ago, the NCEP working group on lipoprotein measurement strongly encouraged the development of a clinically viable assay for direct LDL cholesterol measurement.

It is, however, a fundamental need that the analytical performance of the new proposed methods observes the established qualitative criteria for accuracy and precision to definitively replace both the very time consuming ultracentrifugation based reference method and the quick but inaccurate Friedwald calculation.

In this field the first recommendation by the NCEP working group is intended to provide an LDL measurement that conforms to the epidemiological database relating the LDL cholesterol concentration to cardiovascular risk. In this way the new methods should give results equivalent to those obtained by the Lipid Research Clinics by including in the LDL value the contributions due to other potentially atherogenic particles in addition to LDL.

In general the routine procedures should allow the measurement of LDL cholesterol with a total analytical error not exceeding 12 per cent consistent with limits for accuracy and precision (expressed as CV) of ±4 per cent and lower than 4 per cent respectively for a patient to be correctly classified into the NCEP risk categories.

The Genzyme method was adapted to the Bayer Opera analyser, an open system enabling free programming of nearly all parameter settings with a total turnaround time of 10 minutes. The stability of calibration in the working system was higher than two months on average and a change of reagent lot did not significantly influence it.

In a preliminary experiment the assay precision was evaluated on three human fresh frozen serum pools with different LDL cholesterol concentrations using a modified NCCLS EP5 precision protocol consisting of two replicates of each material over 10 days. Coefficients of variation of 1.3­2.1 per cent were obtained.

The imprecision was also monitored daily in routine practice with the pooled human sera internal quality control material. The mean monthly coefficient of variation was 1.8 per cent showing that long term reproducibility of the method across reagent lots was also quite good.

In both the experimental and practical conditions the assay met the 4 per cent CV NCEP goal for imprecision.

To conclude, the recent development of the direct homogeneous assay for LDL cholesterol determination represents an important advance in laboratory testing now providing a superior method for physicians to confidently diagnose, classify and treat patients according to the NCEP guidelines.

The medical community has recently awaited the availability of a direct test to measure LDL cholesterol. It is hoped that the availability of such a test will encourage more physicians to screen, detect and treat high concentrations of LDL cholesterol in the population, thereby significantly reducing the risk of coronary heart disease.

Enquiry No 12

Mauro Panteghini is a consultant biochemist at the Laboratorio di Biologia e Biochimica, Spedali Civili, Brescia, Italy. This article is based on a presentation at a meeting of the International Federation of Clinical Chemistry, Firenze, Italy, last year. Genzyme Biochemicals is based in West Malling, Kent, UK.





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