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WHAT IS THE BEST EQUATION TO CALCULATE LDL? COMPARISON OF FOUR FORMULAE, INCLUDING THE CLASSIC FRIEDEWALD EQUATION,

WITH DIRECT LDL-C

Martins J1, Olorunju SAS2, Murray LM1, Pillay TS1

1University of Pretoria/NHLS-TAD2Medical Research Council

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Content

• LDL-Cholesterol (LDL-C) LDL-Cholesterol (LDL-C) and coronary heart disease (CHD) risk Laboratory methods to measure LDL-C

• Study Aim Design Evaluation of LDL-C calculations

• Discussion and Conclusion

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Relationship between HDL-C and LDL-C and risk of a coronary heart disease (CHD) event during 4 years of follow-up in the Framingham Study

Taylor A J Eur Heart J Suppl 2006;8:F74-F80

Diagnostic value – LDL-C and Coronary Heart Disease (CHD) risk

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2.57

mmol/l

4.14 5.692.2

1.71.2

0.7

mmol/l

1. Gold standard in routine laboratory practice is ultracentrifugation, but its method is expense and inconvenient

2. Calculations derived from the lipogram

3. Direct measurement of LDL uses a homogenous assay performed on an automated analyser, but remains expensive and shows poor performance if a high triglyceride (TG) values or comorbidities are present

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Measurement of LDL-C in the laboratory

Direct measurement using homogenous assays – 2 selective reagent reaction

PEROXIDASE

Current direct LDL-C reagents not within NCEP Total Error* in diseased patients

*TE criteria = 95% of results ≤13% (HDL) ; ≤12% (LDL-C)

Miller et al. Clin Chem 2010 : 56(6): 977-86

Misclassification rate for CVD risk using direct LDL-C and Friedewald calculated LDL-C per reagent vs reference method

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Direct LDL-C

Calculated LDL-C

van Deventer HE et al. Clin Chem 2011: 57(3): 490-501

Measurement of LDL-C in the laboratory

• Calculations include errors of the various lipid markers in the

lipogram

• The Friedewald equation was developed over 40 years ago is the most widely used formula to calculate LDL-C, however it is

- only used in the fasting state- invalid if TG >4.5 mmol/L <1.13, inaccurate >2 mmol/l- excluded in type III HLP, renal and severe liver disease, diabetes and

metabolic diseases- a derived formula when an LDL-C lower than 1.8 mmo/l was not yet

established as an ideal secondary prevention target for treatment of high-risk patients, thus these levels were not part of the original data set

• About 15 calculations developed since Friedewald, all trying to tackle challenges presented by Friedewald – not all have been validated in large cohorts

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Aim of Study

• Aim:

To establish the most accurate formula for the calculation of LDL-C in hospitalised patients by comparing four formulae Friedewald Chen de Cordova Hattori

to each other and to our direct measurement of LDL-C across various TG, total cholesterol (TC) and HDL-cholesterol (HDL-C) ranges

• A retrospective evaluation of lipid profiles in 14,219 patients from 1 January 2013 to 30 June 2013

• Blood samples were drawn into serum separator tubes by treating physicians in the hospital and clinics served by the National Health Laboratory Services in Pretoria and surrounds

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Study Design

Friedewald FormulaLDL = TC – HDL –(TG/2.2))

vs Ultracentrifuge

Chen FormulaLDL = (TC – HDL) x 0.9 – (TG

x 0.1) vs Roche

Hattori FormulaLDL = 0.94TC – 0.94HDL –

0.19 x TG vs Ultracentrifuge

De Cordova FormulaLDL = 0.7516 (TC – HDL)

vs Wako

Direct LDL-C Daiichi method

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Friedewald WT et al. Clin Chem 1972; 18(6): 499-502de Cordova CM et al. Ann Clin Biochem 2013: 50(Pt 1): 13-9Chen Y et al. Lipids Health Dis 2010;9:52Hattori Y et al. Atherosclerosis 1998;138(2):289-99

2180 outpatients in China – hyperTG

448 subjects in USA (Normal,

Type II/Type IV

hyperlipidaemia

2166 patients with CVD in

Japan

10 664 patients in Brazil with co-

morbidities

A - Friedewald B - De Cordova

C - Chen D - Hattori

Correlation of direct LDL-C with calculated LDL-C in hospitalised patients

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Area under the ROC curve for 4 formulae that calculate LDL-C

The Hattori formula outperformed all formulae over various ranges of lipid values (AUC=0.9097)

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• 574 subjects from the ‘Establishing Reference Intervals for Selected Analytes in South Africa’ study

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A – de Cordova B - Friedewald

Correlation of direct LDL-C with calculated LDL-C in a healthy South African population

Onyenekwu CP et al. Ann Clin Biochem 2014: Jan 21

Bland-Altman plots of direct LDL-C with de Cordova and Friedewald formula in healthy vs hospitalised populations

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B - Friedewald

A – de Cordova

Onyenekwu CP et al. Ann Clin Biochem 2014: Jan 21

Bland-Altman Plot of the Hattori formula vs directly measured LDL-C

The Hattori formula will underestimate LDL-C by only 0.04 mmol/l (vs >0.11 mmol/l with other formulae), demonstrating the best agreement with LDL-C with a level of discrepancy of 0.62 mmol/l.

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Performance of 4 formula to calculate LDL-C across various TG ranges

Formula 0.20 – 29.06 mmol/L 0.20 – 1.02 mmol/L 1.02 – 1.45 mmol/L 1.45 – 2.11 mmol/L 2.11- 5.00mmol/l 5.00 – 29.06mmol/L

ρ Err(N) ρ Err(N) ρ Err(N) ρ Err(N) ρ Err(N) ρ Err(N)

De Cordova 0.93 849.1(5337) 0.97 54.2(1459) 0.98 66.3(1256) 0.97 93.1(1248) 0.94 276.9(1328) 0.92 124.4(135)

Friedewald 0.95 713.6 (5338) 0.97 74.8(1460) 0.98 63.0(1223) 0.97 94.4(1248) 0.96 268.1(1328) 0.95 149.1(135)

Chen 0.95 775.5(5337) 0.98 67.5 (1459) 0.98 74.5 (1200) 0.97 103.8 (1226) 0.95 278.2(1317) 0.94 96.7 (135)

Hattori 0.96 509.4 (5336) 0.98 55.6 (1459) 0.98 61.1 (1256) 0.97 85.9 (1248) 0.96 193.3(1327) 0.95 60.3 (135)

• The de Cordova formula shows favourable correlations at low TG value and is

comparable to Friedewald in the intermediate range• The Friedewald and Chen formulae outperformed de Cordova at high TG values• The Hattori formula indicates best performance across all levels and at extreme lipid values

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• Prediction and management of CVD relies on the accurate measurement of LDL-C

• Gold standard is expensive and direct measurements not recommended in diseased patients

• Calculated LDL-C is the better choice for use in CVD prediction, particularly in dyslipidaemic samples

• Differences found between studies in the application of calculations are likely due to the population studied

• Contribution of LDL aggregates not considered in calculations• The Hattori formula for LDL-C (0.94TC -0.94HDL-C - 0.19 x TG) was

developed to estimate LDL-Apo B and small dense LDL and may be the reason that it is more accurate in patients with cardiovascular co-morbidities and dyslipidaemias

Discussion

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The recently published formula by de Cordova correlates highly with direct measurements of LDL-C, comparing favourably with the Friedewald formula at low TG values

Overall, Friedewald shows better agreement with directly measured LDL-C than de Cordova

However, the Chen and Hattori formulae performed better than the Friedewald

Out of the four formulae evaluated, the Hattori formula appears to be the best for application in hospitalised patients, even at extreme lipid values

Formulae should be validated in local population to establish performance and applicability

Conclusion

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THANK YOU

Acknowledgements:

NHLS – Access to data

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LDL-C equated as a % of non-HDL-C in formulae

• The Chen formula equates LDL-C to 90% of non–HDL-C plus 10% of triglycerides, whereas de Cordova uses 75% of non–HDL-C and Hattori 94% of non-HDL-C

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CVD misclassifications with direct vs calculated LDL-C

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Bland-Altman plots for the 4 formulae

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CVD misclassifications for 2 equations according to HDL-C assay used

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