6to. Congreso Virtual de Cardiología - 6th Virtual Congress of ...

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Index > 6VCC > Epidemiology and Cardiovascular Prevention
Brief Communication
Accuracy of the Friedewald Formula Being in Significant Relation to Total
Cholesterol
Rudolf Gaško, Caio Mauricio Mendes de Cordova*, Slávka Geletková
Railway Hospital and Policlinic Košice, Biostatistic Unit and Dept. of Clinical Biochemistry,
Košice, Slovak Republic, *Departamento de Ciéncias Farmacéuticas, Fundacao Universidade
Regional De Blumenau, Blumenau, Santa Catarina, Brazil
Abstract
Introduction
Numerous epidemiological and clinical studies reported an independent relationship between increases in LDL-cholesterol
(LDL-C)* concentrations and the risk of development of a coronary heart disease. European Fourth Joint Task Force on
Cardiovascular Disease Prevention in Clinical Practice [1] and the US NCEP ATP III also [2] recommends reducing LDL-C to
below certain cut-off points as the primary target of therapy.
The reference method for measuring LDL-C is the ultracentrifugation with beta quantification procedure (BQ), which is not
routinely available in clinical laboratories. The most common approach to determining LDL-C in the clinical laboratory
worldwide is a calculation based on the Friedewald formula [3]. However, there are several well-established limitations to
the Friedewald calculation, which led an expert panel convened by the NCEP to recommending development of accurate
methods for direct determination of LDL-C [4, 5]. Recently, a new generation of homogeneous assays has been introduced
as direct methods, which have been certified by the US CRMLN [4, 5]. Many studies have previously reported that the LDL-C
calculated by the Friedewald formula (FLDL-C) differs from the LDL-C assessed by direct measurement (DLDL-C) or by the
reference method [6]. However, those data showed that differences occurred only over parts of the concentration ranges for
TG, LDL-C and VLDL-C [7-12]. Furthermore, only one study shows a significant relationship to TC [13].
This study compares the effects of both TC and TG concentrations on the difference between the FLDL-C and DLDL-C in
two other populations from Slovakia and Brazil, under conditions allowing for application of the Friedewald formula.
*Non-standard abbreviations: LDL-C, HDL-C, and VLDL-C, LDL-, HDL-, and VLDL-cholesterol; TG, triglyceride; TC, total
cholesterol; NCEP, National Cholesterol Education Program; ATP III, Adult Treatment Panel III; BQ, beta-quantification;
CRMNL, Cholesterol Reference Methods Laboratory Network;
Methods
Košice dataset
For this part of study the blood samples were obtained from 11,331 inpatients and outpatients who visited the Railway
Hospital and Policlinic in Košice, Slovakia, from January 2008 to March 2009. Age range: 18-97 years, mean age: 59
years, males: 53.3%, females: 46.7%. The Institutional Ethics Board approved this examination.
All the persons fasted overnight (for 12 h) before blood collection, and the levels of TC, TG, high-density lipoprotein
cholesterol (HDL-C) and DLDL-C in serum samples were measured on the day of blood collection. Sera with TG level >4.5
mmol/l (1,007 samples, i.e. 8.89% out of 11,331 samples), were excluded. We assessed the total number of 10,324
samples.
TC, TG and HDL-C were measured using enzymatic methods (Pliva Lachema Diagnostika, Brno, Czech Republic). DLDL-C
were measured using a LDL Cholesterol Direct Liquid assay (Pliva Lachema Diagnostika, Brno, Czech Republic), which is
based on the Wako Chemicals method. All measurements were performed on a Olympus AU400 automated clinical
chemistry analyzer (Olympus, Hamburg, Germany).
As regards samples with triglyceride levels < 4,5 mmol/l, the LDL-C level was estimated using the Friedewald formula:
FLDL-C = TC – HDL-C – (TG/2.2).
Blumenau dataset.
This part of the study assessed blood samples of 10,664 patients who sought treatment at Laboratório Santa Isabel de
Análises Clínicas, Blumenau, SC, Brazil, to undergo TC, LDL-C, HDL-C, and TG measurements from January 2000 to
December 2002. Their age ranged from 14 to 93 years (females: 54.8%, males: 45.2%). Blood samples were collected
after a 12- to 14-hour period of fasting, incubated in a hot-water bath for 15 minutes for coagulation, and centrifuged at
2,000g for 5 minutes. The serum was separated and the assays were performed on the day of sample collection. Sera with
TG level >400 mg/dl (340 samples, 3.18% of out 10,664 samples) were excluded. We assessed 10,324 samples.
The measurements of the TG and TC were performed with the following reagents: Triglycerides FS (DiaSys Diagnostic
Systems GmbH & Co. KG, Holzheim, Germany), and Cholesterol (BioSystems S.A., Barcelona, Spain), respectively –
according to the specifications of the manufacturers, in a Spectrum CCX II device (Abbott Diagnostics, Abbott Park, IL,
USA). The HDL-C measurement was performed using a homogeneous method without precipitation with the HDL-C
Immuno FS reagent (DiaSys). The tests were calibrated with the CCX Multicalibrator Set (Abbott), with curves of 3 points.
The LDL-C measurement with the homogeneous method was performed with the reagent LDL-C Select FS (DiaSys), which

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
is based on the Wako Chemicals method.
For samples with triglyceride levels < 400 mg/dL, the LDL-C level was estimated using the Friedewald formula: FLDL-C =
TC – HDL-C – (TG/5).
The patients’ data had been used in study (8) originally.
The %∆DLDL was calculated using the following formula: %∆DLDL=[(FLDL-C – DLDL-C)/DLDL-C]x100, for both datasets.
Statistical analysis
Descriptive statistics, means and standard deviations were calculated, and charts were drawn using Microsoft Office Excel
2007. Paired t-test and correlation analysis was performed using MedCalc, Ver.10.3 (MedCalc Software, Mariakerke,
Belgium).
Results
Košice dataset
The correlation coefficient between the FLDL-C and DLDL-C was 0.94 (p

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Figue 2. Concentrations of triglyceride and total cholesterol affect the percentage difference between
the LDL-C measured directly using a homogeneous assay and that calculated using the Friedewald
formula, data from Kosice.
Blumenau dataset
The correlation coefficient between the FLDL-C and DLDL-C was 0.93 (p

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Figure 3. Concentrations of triglyceride and total cholesterol affect the percentage difference between
the LDL-C measured directly using a homogeneous assay and that calculated using the Friedewald
formula, data from Blumenau.
For comparison, a facsimile of plots based on the data from Korea [13] is shown in Figure 1c, and Figure 4. %∆LDL from all
the three study populations are summarized in Figure 5.
Figure 4. Concentrations of triglyceride and total cholesterol affect the percentage difference between
the LDL-C measured directly using a homogeneous assay and that calculated using the Friedewald
formula, data from Seoul, facsimile from ref. [13].

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Figure 5. Comparison of percentage differences, data from Kosice, Blumenau and Seoul.
Discussion
The European and US guidelines are in agreement as regards the fact that LDL-C lowering comprises the principal target of
lipid treatment. The problem is to achieve a reliable determination of LDL-C (reducing variability of Friedewald LDL-C.)
A major disadvantage related to calculating LDL-C is that the variability is a product of combined variabilities in the three
underlying measurements. The NCEP Expert Panel observed in experienced and well-standardized lipid laboratories that
total analytical variability in calculated LDL-C averaged 4.0%, ranging between 2.7% and 6.8% for LDL-C concentrations
between 2.59-5.83 mmol/l [14]. In routine laboratories, variability appeared to be much higher, e.g. eight survey samples
of the College of American Pathologists (CAP) Comprehensive Chemistry Survey analyzed in more than 1,150 laboratories
gave overall CVs averaging 12% [14]. This CV reflects not only imprecision within laboratories, but also method-to-method
biases from the many different assays used in TC, TG, and HDL-C determinations. The Panel, concluding that many routine
laboratories would not be able to achieve the requisite analytical performance using the Friedewald calculation,
recommended development of more precise direct methods. The TC determinations have the most significant effect on
variability in the calculation [4]. TG values are divided by 2.2 (or 5, for non SI Units) and HDL-C concentrations are
relatively lower, diminishing their impact. Observations from the lipid laboratories as well as from the CAP survey of
routine laboratories suggested that CVs for LDL-C were approximately a double of those for TC [14]. However, increasing
TGs contribute progressively to a higher variability the calculated LDL-C. Adoption of fully automated homogeneous
methods for HDL-C is reality in routine laboratories (including Slovakia and Brazil) in the last years, and addresses the
matter of imprecision, including the contribution to calculated LDL-C; nevertheless, the conclusions of the NCEP Expert
Panel are likely to be still valid.
Priority findings on the direct proportionality of total cholesterol and size %∆LDL, authors Jun et al [13], were confirmed in
both datasets. Both TC and TG represent significant variables affecting the difference between the directly measured and
the calculated LDL-C over the entire range of TC and TG values; the %DLDL increased as TC decreased and TG increased.
Differences in the average value percentage may be due to
- Analytical uncertainty. Analytical uncertainty can not be excluded despite the described good value CV and external
quality assessment. We compared two index methods, not a reference method with an index test.
- Different composition of patients in the sets. It was demonstrated that DM, hepatopathy, nephropathy is substantially
different in FF bias [6]. The divergence structure of both the sets suggests that 8.89% of patients in Kosice had TG higher
then 4.5 mmol/l, but only 3.18% of patients in Blumenau, and TC higher than 6.48 mmol/l – the ratios were 23.84% and
8.04% respectively. Quite on the contrary, TC less than 3.89 mmol/l – the ratios were 4.44% and 13.71% respectively.
- Racial genetic differences [15, 16].
A percentage difference of more than 10% is not surprising as it has already been described. Branchi et al [17] describe
34% of samples from diabetic patients and 26% of samples from non-diabetic patients, but once TG level exceeded 2.26
mmol/l, 45% of samples from diabetic patients and 34% of samples from others differed from measured LDL-C by >10%.
Recent data demonstrate that apolipoprotein B is a better means of measurement of the circulating LDL particle number
concentration and it is also a more reliable indicator of risk than LDL-C, and there is growing support for the idea that
addition of an apo B measurement to the routine lipid panel for assessing and monitoring patients at risk for cardiovascular
disease would enhance patient management [18, 19].

6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Conclusions
It is to be stated that we observed a poor concordance between the calculated and measured LDL cholesterol in three large
populations from Brazil, Slovakia and South Korea, despite a good correlation between the two methods. The accuracy of
the Friedewald formula proves to be in a significant relation to total cholesterol. Further clinical evaluation is required to
verify this finding.
Acknowledgement
This study has been supported by a research grant from the Slovak Society of Cardiology 2008.
References
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6to. Congreso Virtual de Cardiología - 6th Virtual Congress of Cardiology
Publication: September - November/2009
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