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INTRODUCTION
The Korea National Health and Nutrition Examination Survey (KNHANES) comprises a series of studies designed to assess health and nutritional status in the Korean population. KNHANES is one of the most important sources of data for evaluating trends in hyperlipidemia. High-density lipoprotein cholesterol (HDL-C) is a useful marker for evaluating dyslipidemia and therefore was included in the KNHANES as a routine laboratory test. Accurate HDL-C values are essential for the correct use of the KNHANES data and for generating estimates of hypoalphalipoproteinemia burden in the Korean population. HDL-C data are currently available from the KNHANES, but bias in the direct HDL-C assays has limited the utility of these data. Including HDL-C assays by the KNHANES, most commercially available HDL-assays for medical laboratories use homogenous reagents for direct measurement of HDL-C, facilitating automation and improved imprecision over the previously employed precipitation-based HDL-C methods. However, in a recent study, five of the eight examined direct HDL-C assays met the established goals (total error of ≤13% and bias of ≤5%) of the National Cholesterol Education Program (NCEP) working group in samples from normolipidemic individuals, but all assays failed to meet the desired criteria in samples from patients with cardiovascular disease and/or dyslipidemia [1].
The National Health and Nutrition Examination Surveys (NHANES), which determines the prevalence of chronic disease and health conditions in the general noninstitutionalized civilian US population, suggested using calibration equations for correction of the original serum creatinine values in NHANES 1988-1994 and 1999-2000 to estimate kidney function [2]. Calibration equations were derived from comparing serum creatinine values in NHANES data with standard creatinine values measured using an assay traceable to a known gold-standard reference method [2]. For the cystatin C values in the NHANES 1988-1994 and 1999-2002 results, using equations to convert to ERM471/IFCC-traceable cystatin C has been suggested for data users to ensure harmonization [3].
In this study, we aimed to calibrate the original 2008-2015 KNHANES HDL-C values to the Centers for Disease Control and Prevention (CDC) reference method values, using calibration equations based on comparison studies between the HDL-C values obtained by the KNHANES laboratory and those of the CDC reference method [45].
METHODS
1. KNHANES HDL-C assay
From 2008 to 2015, the Seegene Medical Foundation (Seoul, Korea) analyzed all samples for HDL-C testing for the KNHANES using a Hitachi Automatic Analyzer 7600 (Hitachi Co., Tokyo, Japan) and Cholestest N HDL reagent (Sekisui Medical Co., Tokyo, Japan). HDL-C was measured according to the manufacturer's instructions and standard laboratory procedures. Routine internal and external quality control programs, including the Accuracy-Based Lipids (ABL) Survey of the College of American Pathologists (CAP) and the Lipid Standardization Program (LSP) of the CDC (Atlanta, GA, USA), were performed to monitor accuracy and precision.
2. HDL-C comparison studies
We performed three comparison studies (in September 2009, June 2012, and June 2015) using commutable frozen serum (CFS) samples with a wide range of HDL-C values. The CFS samples were prepared and validated according to the CLSI guideline protocol (C37-A: Preparation and Validation of Commutable Frozen Human Serum Pools as Secondary Reference Materials for Cholesterol Measurement Procedure; Approved Guideline) [6]. We used 33 samples in 2009, 19 samples in 2012, and 26 samples in 2015 for the comparison studies. All samples were analyzed in duplicate or triplicate during each run of the study, and one run was conducted per day over two days for each comparison study. The HDL-C CDC reference method values of CFS samples in 2009 were determined by the gas chromatography-isotopic dilution mass spectrometric (IDMS) method at the Lipid Reference Laboratory, Clinical Chemistry Branch, CDC, and those in 2012 and 2015 were measured by the Abell-Kendall (AK) method at the CEQAL Inc. (Vancouver, Canada) of the Cholesterol Reference Method Laboratory Network (CRMLN). The IDMS and AK methods are traceable to the CDC reference method (ultracentrifugation/spectrophotometry method for HDL-C in blood serum) [45]. We compared the HDL-C values quantified by the reference measurement procedure to values measured during the KNHANES (uncalibrated HDL-C values) using Passing-Bablok regression for each comparison study [7].
3. Selection of appropriate calibration equation for each year using accuracy-based external quality assurance data
We determined the appropriate calibration equation to apply for each year based on results of accuracy-based external quality assurance (EQA) programs at the Seegene Medical Foundation, which included the LSP (third quarter 2010 through second quarter 2015) and the ABL Survey (2008 through 2012). We performed a trend analysis of routine HDL-C results to determine the need for data correction with a calibration equation. This analysis was performed according to the year of the sample.
4. Statistical analysis
Statistical analyses were performed by using Excel (Microsoft, Redmond, WA, USA) and Analyse-it (Analyse-it Software Ltd., Leeds, United Kingdom). In the calibration studies, we compared mean HDL-C values using a paired Student's t-test. We used Passing-Bablok regression analysis to obtain the calibration equations [7]. All reported P values were based on 2-sided tests; P values less than 0.05 were considered significant.
RESULTS
Fig. 1 depicts the outline of the present study.
1. KNHANES HDL-C comparison studies
Overall, there was good agreement between the original HDL-C values and reference measurements. Summary statistics and calibration regression equations are presented in Table 1. We derived the calibration regression equations by comparing the uncalibrated HDL-C results with the CDC reference method values using CFS samples. Overall, the mean uncalibrated HDL-C values were higher than the CDC reference method values (P<0.05, all). The degrees of overestimation of uncalibrated HDL-C compared with the CDC reference method values were 4.88 mg/dL in 2009, 1.62 mg/dL in 2012, and 3.13 mg/dL in 2015 (Fig. 2). We examined the intercept and slope obtained by Passing-Bablok regression analyses for each calibration equation [7].
2. Selection of the appropriate calibration equation based on accuracy-based EQA data
We determined the need for HDL-C result correction and selected the appropriate calibration equation for each year through trend analysis of EQA program results (Table 2). The number of samples for each year ranged from 6 to 54. ABL Survey results were derived from a single measurement, and LSP results were the mean values of duplicate measurements. Overall agreement between the original HDL-C values and reference measurements was high. However, since significant positive biases ranging from 2.85% to 9.40% were observed in samples obtained in 2008 to 2015, the correction of the HDL-C values was needed for data obtained in each of these years. We reviewed the calibration equations from our 2009, 2012, and 2015 studies and selected the equation with the least mean difference for each year. The 2009 calibration equation was chosen to correct KNHANES data from 2008, 2009, and 2010. We chose the 2015 equation to correct KNHANES data from 2011 and 2015. Relatively smaller positive biases were observed in 2012, 2013, and 2014; the lowest bias was observed in 2013 (mean bias, 2.85%). For these three years, we used the 2012 equation. We created the following final calibration equations to correct HDL-C values from 2008 to 2015 on the basis of Passing-Bablok regression analysis (Table 3).
3. Distribution of the HDL-C values in KNHANES from 2008 to 2015
The scatter plots of original and calibrated HDL-C values of KNHANES for eight years (2008 to 2015) are presented in Fig. 3. We included all participants aged 30 yr or older in the final analysis. The original values were closer to the reference values once corrected by the calibration equations. Over the 8-yr period, the corrected HDL-C values tended to increase.
DISCUSSION
In this study, the uncalibrated KNHANES HDL-C values from 2008 to 2015 showed substantial positive biases compared with the CDC reference method values. Especially, those from 2008 to 2011 showed biases greater than 5% of the NCEP inaccuracy criterion for lipid testing (5.57-9.40%). The accuracy criterion for the CRMLN, which uses reference methods or designated comparison methods that are rigorously standardized to the CDC reference methods, is ≤1 mg/dL, regardless of the HDL-C level. This degree of biases in HDL-C measurements would yield large differences in hypoalphalipoproteinemia estimates based on KNHANES data. Especially, many individuals' HDL-C values were near the cut-off value of 40 mg/dL for hypoalphalipoproteinemia. Thus, correction of the original KNHANES HDL-C values is needed for appropriate estimation of hypoalphalipoproteinemia prevalence in the Korean population. A small bias in HDL-C values could make a large impact on the calculated prevalence; HDL-C value shift of 2 mg/dL could make a 5-7% shift in the calculated hypoalphalipoproteinemia prevalence in the Korean population according to the previous report of development of trend analysis for lipid profile in the KNHANES [8].
NHANES has been conducted in the United States by the CDC since 1971; corrected serum creatinine, cystatin C, HDL-C, and 25-hydroxyvitamin D values from NHANES data have been reported after correction with calibration equations [23910]. In case of the HDL-C analysis, the heparin-manganese precipitation method and direct immunoassay method for 1999-2000, 2001-2002, and 2005-2006 showed an undesirable bias (>4%) compared with the laboratory's HDL-C quality controls (Solomon Park Research Laboratories, Kirkland, WA, USA) that were assigned values established by the CDC. The HDL-C for 1999-2000, 2001-2002, and 2005-2006 were adjusted by the equation based on the comparison between the quality control HDL-C value associated with each participant sample and its corresponding Solomon Park-assigned HDL-C value [10].
In July 2015, we evaluated the bias of the direct HDL-C assay (Sekisui Medical Co.) of the KNHANES laboratory (Seegene Medical Foundation) compared with the reference method values determined by the CRMLN laboratory at the CEQAL Inc. If the 2015 KNHANES HDL-C values had no bias compared with the CDC reference method values, calibration equations could have been derived from remeasurement of the original KNHANES samples on the basis of a regression analysis between the original values and the remeasured values. However, since the comparison study for 2015 KNHANES HDL-C showed a positive bias of 4.72%, we attempted to determine a calibration equation based on previous comparison studies of HDL-C values of the KNHANES laboratory with the CDC reference method values.
We performed three comparison studies in 2009, 2012, and 2015. Since three calibration equations were only available in our study, we used the accuracy-based EQA results of the KNHANES laboratory to select the appropriate equation for correcting KNHANES HDL-C data in each year from 2008 to 2015. The accuracy-based EQA programs in our study were the LSP of the CDC and the accuracy-based lipid survey of the CAP, whose reference method values were oriented from the CDC reference method performed by the Lipid Reference Laboratory, Clinical Chemistry Branch, CDC or by the CRMLN laboratory at the Northwest Lipid Metabolism and Diabetes Research Laboratories, University of Washington. Since we aimed to calibrate KNHANES HDL-C data to the CDC reference method values, the way to select calibration equation based on the evaluation data for the accuracy-based EQA programs referred to the CDC reference method values could work for our purpose. We calculated mean HDL-C difference (%) using the formula (uncalibrated value - reference value)/reference value×100). The appropriate calibration equation in each year was selected on the basis of the least standard error of mean HDL-C difference corrected by each calibration equation (Table 2).
Our study has some limitations. As at least 24 mL of CFS is needed to obtain a reference value using the CDC HDL-C reference method [456], we could not ensure a sufficient number of samples for the comparison studies (33 samples in 2009, 19 samples in 2012, and 26 samples in 2015, respectively). The CLSI guideline EP09-A3 recommends the use of at least 40 samples to estimate bias to improve confidence in the statistical estimates and increase the opportunity to incorporate the effects of unexpected interfering substances (individual idiosyncratic biases) [11]. Therefore, the small number of samples for comparison may be a limitation to deriving adequate calibration equations for data correction. Including the limitation of small sample number in the comparison, our suggestion of using calibration equations based on the comparison studies for correction should be considered as one of the ways to calibrate KNHANES data. Further studies would be needed to find a better way.
In summary, our results provide a basis for HDL-C data correction from the KNHANES and provide a calibration equation to ensure standardization. The recommended equation for correcting the original HDL-C values obtained from KNHANES in 2008-2015 can be used to eliminate bias in the originally reported measurements that resulted from drift in the calibration of the measurement procedures. However, since the need to correct biases could be changed by the characteristics of research topics, the decision to convert the KNHANES data using these equations should be decided on an individual basis in each study.
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