Performance of Four Homogeneous Direct Methods for LDL-Cholesterol

Abstract Background: Homogeneous LDL-cholesterol methods from Genzyme, Reference Diagnostics, Roche, and Sigma were evaluated for precision, accuracy, and specificity for LDL in the presence of abnormal lipoproteins. Methods: Each homogeneous method was performed by a Roche/Hitachi 911 according to the vendors’ instructions, and the results were compared with the β-quantification reference method. We measured precision over 20 days using quality-control and frozen serum specimens. Sera from 100 study participants, including 60 with hyperlipidemias, were assayed by each method. Accuracy was evaluated from regression and total error analysis. Specificity was evaluated from the bias (as a percentage) vs concentration of triglycerides. Results: The total CV was <2% for all methods. Regression slope and intercept (with 95% confidence intervals) were as follows: Genzyme, 0.955 (0.92 to 0.99) and 30.3 (−12 to 73) mg/L; Reference Diagnostics, 0.975 (0.93 to 1.02) and −8 (−63 to 47) mg/L; Roche, 1.067 (1.02 to 1.11) and −101 (−161 to −42) mg/L; and Sigma, 0.964 (0.91 to 1.02) and 164 (89 to 239) mg/L. The percentages of individual results with >12% bias were as follows: Genzyme, 8.0%; Reference Diagnostics, 11.0%; Roche, 10.0%; and Sigma, 30.0%. Total error calculated from mean systematic bias and all-sources random bias was as follows: Genzyme, 12.6%; Reference Diagnostics, 16.5%; Roche, 41.6%; and Sigma, 38.3%. Slopes of bias (as a percentage) vs triglycerides were P <0.001 for all methods except the Roche method, which was P = 0.094. Conclusions: The evaluated methods show nonspecificity toward abnormal lipoproteins, thus compromising their ability to satisfy the National Cholesterol Education Program goal for a total error of <12%. These homogeneous LDL-cholesterol results do not improve on the performance of LDL-cholesterol calculated by the Friedewald equation at triglyceride concentrations <4000 mg/L.

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Analytical and Clinical Evaluation of Two Homogeneous Assays for LDL-Cholesterol in Hyperlipidemic Patients

Clinical Chemistry ◽

10.1093/clinchem/46.8.1121 ◽

2000 ◽

Vol 46 (8) ◽

pp. 1121-1131 ◽

Cited By ~ 35

Author(s):

Margarita Esteban-Salán ◽

Amada Guimón-Bardesi ◽

Jesús María de la Viuda-Unzueta ◽

María Nerea Azcarate-Ania ◽

Pilar Pascual-Usandizaga ◽

...

Keyword(s):

Ldl Cholesterol ◽

Treatment Guidelines ◽

Clinical Performance ◽

Total Error ◽

Direct Methods ◽

Lipid Lowering ◽

Serum Samples ◽

Wide Range ◽

Friedewald Formula ◽

Homogeneous Assays

Abstract Background: LDL-cholesterol (LDL-C) concentrations are the primary basis for treatment guidelines established for hyperlipidemic patients. LDL-C concentrations are commonly monitored by means of the Friedewald formula, which provides a relative estimation of LDL-C concentration when the triglyceride concentration is <2000 mg/L and there are no abnormal lipids. The Friedewald formula has several limitations and may not meet the current total error requirement of <12% in LDL-C measurements. Methods: We evaluated the analytical and clinical performance of two direct methods (Roche and Wako) by analyzing 313 fresh serum samples obtained from dyslipidemic patients in a lipid clinic and comparing them with modified β-quantification. Results: Both homogeneous assays displayed excellent precision (CV <2%). The Roche method showed a mean total error of 7.72%, and the Wako method showed a mean total error of 4.46% over a wide range of LDL-C concentrations. The Roche method correlated highly with the modified β-quantification assay (r = 0.929; y = 1.052x − 168 mg/L; n = 166) and showed a bias of −4.5% as a result of the assigned standard value. The Wako method also correlated highly with β-quantification (r = 0.966; y = 0.9125x + 104.8 mg/L; n = 145) without significant bias. The Roche method correctly classified 97% of patients with triglycerides <2000 mg/L, 75% of patients with type IIb hyperlipemia (HPL), and 84% of patients with type IV HPL based on the cutpoints of 1300 and 1600 mg/L, compared with 98%, 78.4%, and 89%, respectively, for the Wako method. In dysbetalipoproteinemic patients, both methods have a 30% mean positive bias compared with β-quantification. Conclusions: Both direct methods can be a useful alternative when ultracentrifugation is not available for the diagnosis and control of lipid-lowering medication for patients with mixed HPL, but not for patients with type III hyperlipidemia.

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Measurements for 8 Common Analytes in Native Sera Identify Inadequate Standardization among 6 Routine Laboratory Assays

Clinical Chemistry ◽

10.1373/clinchem.2013.220376 ◽

2014 ◽

Vol 60 (6) ◽

pp. 855-863 ◽

Cited By ~ 20

Author(s):

Hedwig C M Stepman ◽

Ulla Tiikkainen ◽

Dietmar Stöckl ◽

Hubert W Vesper ◽

Selvin H Edwards ◽

...

Keyword(s):

Uric Acid ◽

Ldl Cholesterol ◽

Peer Group ◽

Clinical Chemistry ◽

Total Error ◽

Random Access ◽

Reference Method ◽

Hdl Cholesterol ◽

Serum Samples ◽

Fresh Frozen

Abstract BACKGROUND External quality assessment (EQA) with commutable samples is essential for assessing the quality of assays performed by laboratories, particularly when the emphasis is on their standardization status and interchangeability of results. METHODS We used a panel of 20 fresh-frozen single-donation serum samples to assess assays for the measurement of creatinine, glucose, phosphate, uric acid, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. The commercial random access platforms included: Abbott Architect, Beckman Coulter AU, Ortho Vitros, Roche Cobas, Siemens Advia, and Thermo Scientific Konelab. The assessment was done at the peer group level and by comparison against the all-method trimmed mean or reference method values, where available. The considered quality indicators were intraassay imprecision, combined imprecision (including sample–matrix interference), bias, and total error. Fail/pass decisions were based on limits reflecting state-of-the-art performance, but also limits related to biological variation. RESULTS Most assays showed excellent peer performance attributes, except for HDL- and LDL cholesterol. Cases in which individual assays had biases exceeding the used limits were the Siemens Advia creatinine (−4.2%), Ortho Vitros phosphate (8.9%), Beckman Coulter AU triglycerides (5.4%), and Thermo Scientific Konelab uric acid (6.4%), which lead to considerable interassay discrepancies. Additionally, large laboratory effects were observed that caused interlaboratory differences of >30%. CONCLUSIONS The design of the EQA study was well suited for monitoring different quality attributes of assays performed in daily laboratory practice. There is a need for improvement, even for simple clinical chemistry analytes. In particular, the interchangeability of results remains jeopardized both by assay standardization issues and individual laboratory effects.

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Limitations of Direct Methods and the Reference Method for Measuring HDL and LDL Cholesterol

Clinical Chemistry ◽

10.1373/clinchem.2010.159483 ◽

2011 ◽

Vol 57 (7) ◽

pp. 1081-1083 ◽

Cited By ~ 3

Author(s):

Ernst J Schaefer ◽

Seiko Otokozawa ◽

Masumi Ai

Keyword(s):

Ldl Cholesterol ◽

Reference Method ◽

Direct Methods

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Seven Direct Methods for Measuring HDL and LDL Cholesterol Compared with Ultracentrifugation Reference Measurement Procedures

Clinical Chemistry ◽

10.1373/clinchem.2009.142810 ◽

2010 ◽

Vol 56 (6) ◽

pp. 977-986 ◽

Cited By ~ 169

Author(s):

W Greg Miller ◽

Gary L Myers ◽

Ikunosuke Sakurabayashi ◽

Lorin M Bachmann ◽

Samuel P Caudill ◽

...

Keyword(s):

National Cholesterol Education Program ◽

Ldl Cholesterol ◽

Direct Method ◽

Total Error ◽

Direct Methods ◽

Hdl Cholesterol ◽

Individual Sample ◽

Serum Samples ◽

Reference Measurement ◽

The Difference

Abstract Background: Methods from 7 manufacturers and 1 distributor for directly measuring HDL cholesterol (C) and LDL-C were evaluated for imprecision, trueness, total error, and specificity in nonfrozen serum samples. Methods: We performed each direct method according to the manufacturer’s instructions, using a Roche/Hitachi 917 analyzer, and compared the results with those obtained with reference measurement procedures for HDL-C and LDL-C. Imprecision was estimated for 35 runs performed with frozen pooled serum specimens and triplicate measurements on each individual sample. Sera from 37 individuals without disease and 138 with disease (primarily dyslipidemic and cardiovascular) were measured by each method. Trueness and total error were evaluated from the difference between the direct methods and reference measurement procedures. Specificity was evaluated from the dispersion in differences observed. Results: Imprecision data based on 4 frozen serum pools showed total CVs <3.7% for HDL-C and <4.4% for LDL-C. Bias for the nondiseased group ranged from −5.4% to 4.8% for HDL-C and from −6.8% to 1.1% for LDL-C, and for the diseased group from −8.6% to 8.8% for HDL-C and from −11.8% to 4.1% for LDL-C. Total error for the nondiseased group ranged from −13.4% to 13.6% for HDL-C and from −13.3% to 13.5% for LDL-C, and for the diseased group from −19.8% to 36.3% for HDL-C and from −26.6% to 31.9% for LDL-C. Conclusions: Six of 8 HDL-C and 5 of 8 LDL-C direct methods met the National Cholesterol Education Program total error goals for nondiseased individuals. All the methods failed to meet these goals for diseased individuals, however, because of lack of specificity toward abnormal lipoproteins.

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Optimization of β-Quantification Methods for High-Throughput Applications

Clinical Chemistry ◽

10.1093/clinchem/47.4.712 ◽

2001 ◽

Vol 47 (4) ◽

pp. 712-721 ◽

Cited By ~ 19

Author(s):

Thomas G Cole ◽

Constance A Ferguson ◽

David W Gibson ◽

William L Nowatzke

Keyword(s):

Ldl Cholesterol ◽

Total Error ◽

Hdl Cholesterol ◽

Basic Research ◽

Turnaround Time ◽

Cholesterol Concentration ◽

Target Values ◽

Bottom Fraction ◽

Clinically Significant ◽

Method Accuracy

Abstract Background: Risk of cardiovascular disease is assessed, in part, by laboratory measurement of the concentrations of several lipoproteins. β-Quantification is a method of lipoprotein measurement that uses ultracentrifugation to partially separate lipoprotein classes. Although β-quantification is used largely in clinical and basic research, methods have not been described to allow the analysis of a large number of small-volume specimens with a short turnaround time. We report two variations of the traditional 5-mL method used by the Lipid Research Clinics Program that overcome these shortcomings. Methods: Two lower-volume modifications of the traditional 5-mL β-quantification method were developed. The methods used either 1 or 0.23 mL of specimen and required substantially less time for analysis (20 and 6 h, respectively) than the 5-mL method (2.5 days). The goal was to develop ultracentrifugation methods such that the concentration of cholesterol in the bottom fraction, from which LDL-cholesterol concentration is calculated, agreed with the 5-mL method. Fresh serum specimens (n = 45) were analyzed by the three methods to determine comparability of the methods based on the recovery of cholesterol in the bottom fraction after ultracentrifugation. To evaluate intrarun precision, replicate specimens (n = 17) were analyzed in a single run for each method. This experiment also evaluated how quickly the fractions would remix after separation by ultracentrifugation. For the 1-mL method, accuracy of the measurement of LDL- and HDL-cholesterol concentrations and the interrun precision were established by analysis of frozen serum specimens provided by the CDC, which established target values for the pools using reference methods. Results: No clinically significant differences in cholesterol concentrations in the bottom fraction were observed for the 1- and 0.23-mL methods, which had mean biases of 0.8% and 1.5% relative to the 5-mL method, respectively. Intra- and interrun variability was acceptable for each method, e.g., <1.8% for cholesterol in the bottom fraction. Ultracentrifuged specimens were stable for at least 4 h with no evidence of contamination of cholesterol in the bottom fraction. For comparison specimens provided by the CDC, the 1-mL method met the accuracy and precision goals of the National Cholesterol Education Program for the measurement of HDL- and LDL-cholesterol concentrations (goals: total error <13% and <12%, respectively), with total errors of 6.45% and 5.43%, respectively. Conclusions: Both the 1- and 0.23-mL β-quantification methods are suitable substitutes for the traditional 5-mL method for use in clinical and basic research for the determination of LDL-cholesterol concentration. Both methods provide much higher throughput and require substantially less specimen volume. The 0.23-mL method can be performed in 1 day, but it is slightly less precise than the 1-mL method. In our laboratory setting, as many as 80 specimens are routinely processed per day using the 1-mL method.

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Validation of a Proposed Novel Equation for Estimating LDL Cholesterol

Clinical Chemistry ◽

10.1373/clinchem.2014.227710 ◽

2014 ◽

Vol 60 (12) ◽

pp. 1519-1523 ◽

Cited By ~ 38

Author(s):

Jeffrey W Meeusen ◽

Alan J Lueke ◽

Allan S Jaffe ◽

Amy K Saenger

Keyword(s):

Patient Outcomes ◽

Ldl Cholesterol ◽

Reference Method ◽

Routine Clinical Practice ◽

The Novel ◽

Primary And Secondary Prevention ◽

Friedewald Equation ◽

Novel Method ◽

Improve Patient ◽

Independent Cohort

Abstract BACKGROUND Aggressive LDL cholesterol (LDL-C)-lowering strategies are recommended for primary and secondary prevention of cardiovascular events. A newly derived equation for LDL-C estimation was recently published that addressed limitations in the commonly used Friedewald LDL-C calculation method. The novel method was reported to classify patients with superior concordance to measured LDL-C compared to the Friedewald method, particularly in patients with LDL-C <70 mg/dL. METHODS We evaluated the performance of the novel method within an independent cohort of 23 055 patients with LDL-C measured by the gold standard β-quantification reference method. RESULTS Overall Friedewald underestimated and the novel method overestimated measured LDL-C. Both estimations significantly deviated from the reference method when LDL-C was <70 mg/dL. Overall, the Friedewald and novel calculations correctly classified 77% and 78% of patients, respectively. The largest discrepancy in classification was observed in individuals with measured LDL-C <70 mg/dL. For this group the novel calculation would reclassify 8.7% of patients as >70 mg/dL compared to the Friedewald equation. CONCLUSIONS We compared both novel and Friedewald estimated LDL-C against the LDL-C reference method; in contrast, the prior study relied on validation of a subset of samples by β-quantification to allow the use of the vertical autoprofile method for LDL-C measurement. We conclude that the novel method has some benefits but it is unclear whether improvements over the Friedewald calculation are substantive enough to justify making the change in routine clinical practice and to improve patient outcomes.

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Improved Specificity of a New Homogeneous Assay for LDL-Cholesterol in Serum with Abnormal Lipoproteins

Clinical Chemistry ◽

10.1373/clinchem.2005.053983 ◽

2006 ◽

Vol 52 (5) ◽

pp. 886-888 ◽

Cited By ~ 8

Author(s):

Yasumasa Iwasaki ◽

Hiroyuki Matsuyama ◽

Nobuo Nakashima

Keyword(s):

Clinical Practice ◽

Obstructive Jaundice ◽

Ldl Cholesterol ◽

Performance Characteristics ◽

New Method ◽

Assay Performance ◽

Basic Performance ◽

Homogeneous Method ◽

Homogeneous Assay ◽

Improved Performance

Abstract Background: Although a homogeneous assay for serum LDL-cholesterol (LDL-C) has become a routine clinical procedure, problems remain in assay performance characteristics. Methods: We examined the performance of a recently developed automated homogeneous assay (New-Daiichi assay) for serum LDL-C and compared the results with those obtained by the current homogeneous method (Denka-Seiken assay) or by ultracentrifugation as a control. Results: The New-Daiichi assay showed satisfactory basic performance characteristics such as reproducibility, linearity, and stability. There was no interference in the assay by various substances examined. The LDL-C values obtained with this method correlated well with those obtained by ultracentrifugation. In samples from patients with obstructive jaundice, both methods detected cholesterol from abnormal lipoproteins (such as lipoprotein-X and -Y), but the New-Daiichi assay was less reactive and more specific for LDL-C. Conclusion: The new method has improved performance for the accurate measurement of LDL-C in clinical practice.

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Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method

Clinical Chemistry ◽

10.1093/clinchem/44.6.1242 ◽

1998 ◽

Vol 44 (6) ◽

pp. 1242-1250 ◽

Cited By ~ 71

Author(s):

Nader Rifai ◽

Elizabeth Iannotti ◽

Kristen DeAngelis ◽

Terence Law

Keyword(s):

Dextran Sulfate ◽

Ldl Cholesterol ◽

Clinical Performance ◽

Total Error ◽

Analytical Performance ◽

Medical Decision ◽

Serum Samples ◽

Predictive Values ◽

Clinical Laboratories ◽

Wide Range

Abstract LDL-cholesterol (LDL-C) concentration is currently determined in most clinical laboratories by the Friedewald calculation. This approach has several limitations and may not meet the current total error requirement in LDL-C measurement of ≤ 12%. We evaluated the analytical and clinical performance of the direct N-geneous LDL-C assay (Equal Diagnostics). The N-geneous method correlated highly with the modified beta-quantification assay (r = 0.95; y = 0.91x + 70.6 mg/L; n = 199), showed no significant effect of increased triglyceride or other common interferants, and performed adequately in serum samples from nonfasting individuals. This assay demonstrated a mean total error of 6.75% over a wide range of LDL-C concentrations. In addition, at the medical decision cutoff points, this LDL-C assay showed positive predictive values of 78–95% and negative predictive values of 84–99%. We conclude that the N-geneous LDL-C meets the currently established analytical performance goals and appears to have a role in the diagnosis and management of hypercholesterolemic patients.

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