Direct measurement of lipid fractions provides superior accuracy and precision in lipid profile tests (lpt) over calculated formulas
Editors:
- Dr. Praful B. Godkar (Ph.D)
Eminent Author, Medical Biochemist and Scientist, Technical Education consultant. AGD Biomedicals (Pvt) LTD. - Dr. Gauri Kulkarni MD (Pathology)
Vice President, AGD Biomedicals (Pvt) LTD.
A Lipid Profile Test (LPT) is mainly performed to evaluate cardiovascular health and to estimate the likelihood of developing clinical conditions such as atherosclerosis, heart disease, or stroke. LPT is extremely useful to identify silent risks before they lead to life-threatening events(1). The prevailing economic practice is to utilize the Friedewald Equation (FE) for determining values including LDL, HDL, and VLDL, as well as ratios such as Total Cholesterol/HDL and LDL/HDL(1). While the commonly utilized FE allows for the estimation of LDL, HDL, and VLDL, it possesses inherent limitations(1) Refer answer to Q2. AGD Biomedical PVT LTD, Mumbai, India, provides economical specialized homogeneous enzymatic reagents for direct measurement of lipid fractions, which provide superior accuracy in LPT over calculated formulas, with TEa (Total Allowable Error) and TE (Total Error) calculations as per National Cholesterol Education Program (NCEP) guidelines for accuracy and precision(1,2).
Q1. What is the Friedewald equation?
ANS(1): The Friedewald equation is used to determine low-density lipoprotein LDL-cholesterol (LDL-c), and VLDL-cholesterol, using serum total cholesterol, HDL-cholesterol and serum triglycerides values by means of the following equation:
VLDL-C, mg/dl = Triglycerides/5
LDL-C, mg/dl = Total cholesterol – HDL-C – Triglycerides/5
Q2. What are the drawbacks of Friedewald equation?
ANS(1): The Friedewald equation, which calculates LDL as (Total Cholesterol – HDL – [Triglycerides/5]), can become inaccurate when triglyceride levels are high (>400 mg/dL) or when LDL levels are very low.
NOTE
Research studies show that direct measurement of lipid fractions with homogeneous enzymatic reagents is more accurate than calculated formulas like the Friedewald equation.
Q3. What is the clinical significance of Lipid Profile Tests performed with high accuracy and precision using specialized homogeneous enzymatic reagents of AGD Biomedicals PVT LTD?
ANS(1): Refer Table 1.
Table 1: Clinical Significance of Serum Lipid Profile Tests
| Component | Clinical Significance | REFERENCE RANGE (Adults) |
| Total Cholesterol | Cardiovascular risk estimation. | < 200 mg/dL |
| LDL (Bad cholesterol) | High levels indicate risk for plaque buildup in arteries, leading to atherosclerosis. | < 100 mg/dL |
| HDL (Good cholesterol) | Prevent acceleration of atherosclerosis by carrying excess cholesterol back to the liver for removal. Good for normal functions of the heart | > 60 mg/dL |
| Triglycerides | Elevated levels are associated with obesity, Type 2 diabetes, atherosclerosis, and higher risk to heart health. | < 150 mg/dL |
| VLDL | High values lead to risk of arterial plaque formation. | 2 – 30 mg/dL |
NOTE(1)
(A) Total allowable error (TEa) for lipids, based on NCEP guidelines, requires total cholesterol to be within ±9%, HDL-C within ±13%, LDL-C within ±12% and, and triglycerides within ±15%. These limits are crucial for ensuring accurate cardiovascular risk assessment, with recommended precision (CV) and bias both below 3% for total cholesterol, LDL and below 5% for triglycerides and VLDL respectively. Exceeding these limits, such as a total error (Bias + CV) > 9% for cholesterol, renders patient results unreliable.
(B) TE typically stands for Total Error. Laboratory quality is often managed through TEa (Total Allowable Error) and TE (Total Error) calculations.
(C) For laboratory measurements, Total Quality Management (TQM) related to the sigma performance of a method is calculated from the imprecision as indicated by the standard deviation (SD) or the coefficient of variation (CV), and inaccuracy (bias) observed for a method and the allowable total error (TEa) for the test. Hence, Sigma = TEa – bias / CV.
EXAMPLE
In the Total Quality Management (TQM) of a particular test and related reagents, Sigma metric system of TQM from 6.0 to 3.0 represents the range from “best case” to “worst case”. For example, for a cholesterol test with a CLIA criterion of 10%, method bias of 1%, and method CV of 2%, the sigma metric is 4.5 [10 – 1.0 / 2.0], which is good. If the method had a CV of 3% and a bias of 3.0%, the Sigma metric is 2.33. Methods with sigma performance of less than 3 are not acceptable(1).
Q4. What is the principle on which HDL-cholesterol determination is based using AGD Biomedicals PVT LTD specialized reagents?
ANS(1): Determination of HDL-cholesterol is based on Immunoinhibition method and can be performed manually as well as on autoanalyzers and includes two reagents. The first reagent contains an antibody to human apo B-100. It reacts with the apo B-100 containing lipoproteins such as chylomicrons, VLDL, and LDL and blocks the reaction of cholesterol contents of chylomicrons, VLDL, and LDL with enzymes present in the second reagent (cholesterol esterase, cholesterol oxidase and peroxidase). Cholesterol esterase, and cholesterol oxidase enzymes then act on esterified and free cholesterol of HDL fraction with peroxidase and the O. D. of color produced at the end of the reaction is directly proportional to the concentration of serum HDL-cholesterol.
Q5. What is the principle on which LDL-cholesterol determination is based using AGD Biomedicals PVT LTD specialized reagents?
ANS(1): Two reagents are used for this method. The initial reagent is formulated with the detergent calixarene, facilitating the transformation of serum LDL into a soluble complex. Cholesterol esters of HDL-cholesterol and VLDL-cholesterol are hydrolyzed by cholesterol esterase and cholesterol oxidase, present in the reagent respectively. Hydrazine converts the accessible cholesterol to cholestenone hydrazone. The second reagent contains deoxycholate, breaks the LDL-calixarene complex and releases LDL-cholesterol. LDL-cholesterol reacts with the cholesterol esterase, a dehydrogenase and ß-NAD, and the end product ß-NADH is measured at 340 nm. The concentration of ß-NADH is directly proportional to serum LDL-cholesterol.
CASE 1
A routine blood examination for a 38-year-old male executive revealed the following lipid profile results:
LIPID PROFILE TESTS
| PARAMETER | RESULT | REFERENCE VALUES |
| Blood glucose fasting | 95 mg/dl | 70–110 mg/dl |
| Serum total cholesterol | 287 mg/dl | 150–250 mg/dl |
| Serum triglycerides | 436 mg/dl | 10–190 mg/dl |
| Serum LDL-cholesterol | 175 mg/dl | < 130 mg/dl |
| Serum VLDL | 261 mg/dl | 2–38 mg/dl |
| Serum HDL-cholesterol | 53 mg/dl | > 55 mg/dl |
| Total serum cholesterol / HDL-cholesterol | 5.2 | < 5 |
| LDL-cholesterol / HDL-cholesterol | 3.3 | 0.5–3.0 |
INTERPRETATION(1)
This person is not diabetic. Increased values of serum total cholesterol, LDL, triglycerides and VLDL indicate Hyperlipidemia of 2b Phenotype according to Fredrikson’s hyperlipidemia classification. It indicates +++ atherogenicity. Total serum cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratios indicate increased atherogenic risk and ischemic heart disease.
NOTE
(A) In spite of very high values of serum triglycerides (>400 mg/dl), AGD Biomedical’s specialized homogeneous enzymatic reagents used for direct determination of serum lipid fractions could yield accurate and precise results.
(B) All homogeneous assays for LDL-C and HDL-C should meet the National Cholesterol Education Program (NCEP) requirements in terms of coefficient of variation, and TEcom in both non-diseased and diseased subjects. LDL-C and HDL-C values measured by the homogeneous assays should be in good agreement with those measured by the Reference Measurement Procedures (RMPs), in both fasting and postprandial samples. The TEcom and TEECA values showed strong agreement between postprandial and fasting samples.
(C) Tecom and TEECA values are used in clinical biochemistry to evaluate the reliability of homogeneous assays (e.g., LDL-C/HDL-C) compared to Reference Measurement Procedures (RMPs).
TEcom (Total Error component): Represents the total error of the commercial method.
TEECA (Total Error Estimating Component of Accuracy): A calculation used to assess the total allowable error.
References
(1) Godkar PB, Godkar DP. Textbook of Medical laboratory technology (4th edition, 2024), Chapters 6 and 16. Bhalani Publishers, Mumbai. India.
(2) Takashi Miida, Kunihiro Nishimura, Satoshi Hirayama, Yoshihiro Miyamoto, Masakazu Nakamura, Daisaku Masuda, Shizuya Yamashita, Masaji Ushiyama, Toshiaki Komori, Naohisa Fujita, Shinji Yokoyama and Tamio Teramoto. Department of Clinical Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo. Homogeneous Assays for LDL-C and HDL-C are Reliable in Both the Postprandial and Fasting State. J Atheroscler Thromb, 2017; 24: 583-599. ttp://doi.org/10.5551/jat.40006
