Evaluation of Homogeneous High-Density Lipoprotein Cholesterol Assay on a BM/Hitachi 747–200 Analyzer

¹ Department of Chemistry, The Permanente Medical Group, Inc., Regional Laboratory, Berkeley, CA 94710-1798 and
² Division of Research, Kaiser Permanente Medical Care Program, Oakland, CA 94612-3429;
^a address for correspondence: 1725 Eastshore Hwy., Berkeley, CA 94710-1798

Because of the inverse correlation that exists between serum HDL-C concentration and risk of atherosclerotic disease (1), monitoring of high-density lipoprotein cholesterol (HDL-C) in serum is clinically important. In 1993, the National Cholesterol Education Program Adult Treatment Panel II (NCEP ATP II) revised its guidelines for diagnosis and treatment of hypercholesterolemia in adults to include HDL-C measurement at the initial screening stage when total cholesterol is measured (2). The enhanced role of HDL-C in clinical practice increases the need for reliable and readily performable HDL-C measurement. Most routine laboratories use a two-step method: chemical precipitation of lipoproteins containing apoprotein B, then quantification of HDL as cholesterol remaining in the supernate. Such precipitation-based methods are time-consuming, labor-intensive, require relatively large volumes of serum, and cannot be fully automated. Recently, several methods of direct HDL-C measurement have become commercially available, including the use of magnetically responsive particles with polyanion-metal combinations (3), the use of polyethylene glycol (PEG) with antibodies against apoprotein B and apoprotein CIII (4)(5), and the use of PEG-modified enzymes and sulfated -cyclodextrin (6)(7). Okazaki et al. (8) also recently proposed the use of high-performance liquid chromatography (HPLC) as a tool to compare different methods for HDL-C. The goal of this study was to evaluate three assays for homogeneous HDL-C on BM/Hitachi 747–200 Automatic Analyzer (Boehringer Mannheim Corp.): the direct HDL-cholesterol reagent kit (Boehringer Mannheim Corp.), the N-geneous^TM HDL cholesterol reagent kit (Genzyme Diagnostics), and EZ-HDL^TM cholesterol reagent kit (Sigma Diagnostics). For each HDL-C assay, the reagent kit contains Reagent 1 and Reagent 2; Reagent 1 is liquid, and Reagent 2 is lyophilized. Reaction principles of these three methods are quite different; the direct-HDL uses PEG-modified cholesterol esterase and cholesterol oxidase as well as sulfated -cyclodextrin to provide selective determination of HDL-C in serum; the N-geneous^TM HDL uses polyanions and synthetic polymers to aggregate the VLDL, LDL, and chylomicron into complexes, after which a detergent selectively releases HDL cholesterol to react with cholesterol enzymes; and the EZ-HDL^TM uses an immunoinhibition enzymatic method that binds lipoproteins other than HDL with anti-human ß-lipoprotein antibodies to form antigen-antibody complexes so that cholesterol esterase and cholesterol oxidase react only with HDL-C.

Imprecision was evaluated by using the NCCLS EP5-T protocol (9); control material (Sigma Diagnostics) was used to test for low and medium HDL-C concentrations, and one human serum pool was used to test for high HDL-C concentration (we divided serum into 20 aliquots and stored it at -20 °C). Each homogeneous HDL-C assay was performed in two analyses per day for 20 days. Assay bias was calculated as the test method result minus the CDC Reference Method (10) result from three different HDL-C concentrations (267, 440, and 588 mg/L) of pool sera, which were provided by the Centers for Disease Control and Prevention. Total imprecision is shown in Table 1 . The bias was -5.5% to 1.9% for the direct HDL-C, -7.0% to 1.5% for the N-geneous^TM HDL-C, and 3.7% to 7.9% for the EZ-HDL^TM-C. Total imprecision and bias for the three homogeneous HDL-C assays were acceptable according to NCEP performance goals: total imprecision 6% and bias less than or equal to ±10%. To assess linearity of the assays, we used a high-concentration (2000 mg/L), serum-based HDL-C sample (Biocell Laboratories, Inc.) and diluted it with 9 g/L NaCl to produce concentrations corresponding to 20%, 40%, 60%, 80%, and 90% of the original concentration. Linearity was established up to 1800 mg/L for the direct HDL-cholesterol reagent kit (r = 1.00, slope = 0.99), up to 1600 mg/L (r = 1.00, slope = 0.99) for the N-geneous^TM HDL cholesterol reagent kit, and up to 2000 mg/L (r = 1.00, slope = 0.99) for the EZ-HDL^TM cholesterol reagent kit. We compared these homogeneous HDL-C assays with our present method (phosphotungstic acid without ion precipitation) by analyzing patient serum samples in parallel and in five separate analyses within 20 days. All serum samples were originally submitted to our laboratory for routine lipid panel screening from 23 Northern California Kaiser Permanente medical centers and medical offices. No samples were obtained solely for this study. Serum samples were stored at 2–8°C and were analyzed within 5 days. Total cholesterol concentration was measured by enzymatic method, and triglyceride concentration was measured by the glycerol phosphate oxidase method. Mean (range) of total cholesterol concentration was 2240 mg/L (1410–3290 mg/L) and was 2410 mg/L (330–7930 mg/L) for triglyceride concentration. The Pearson correlation coefficient and Deming regression analysis were used to compare methods. Results obtained using all three homogeneous HDL methods correlated highly with those of our current method (r = 0.99). Regression equations were: y = 1.069x 2.9 (n = 240) for the direct HDL-C assay, 0.95x 7.8 (n = 190) for the N-geneous^TM HDL-C assay, and 0.98x 6.6 (n = 120) for the EZ-HDL^TM-C assay.

Possible interference with these homogeneous HDL-C assays by lipemia, hyperbilirubinemia, and hemolysis was investigated. We used the procedure of Glick et al. (11) to supplement separate serum pools with graded concentrations of fresh hemolysate (Hb 10 000 mg/L), Intralipid^TM (Kabipharmacia, Inc.) containing triglyceride 32 000 mg/L, and bilirubin (Pfanstiehl Laboratories, Inc.) 540 mg/L. Recovery results were decreased by hemolysis: 93.4% to 100% for the direct HDL-C assay, 87.5% to 100% for the N-geneous^TM HDL-C assay, and 90% to 100% for the EZ-HDL^TM-C assay. No statistically significant interference (recovery results: 91.5% to 104%) from bilirubin concentrations up to 540 mg/L was detected. Negative interference (a decrease of 10% from the original value) by artificial lipemia was observed at a triglyceride concentration of 21 270 mg/L for both the N-geneous^TM HDL-C and EZ-HDL^TM-C assays, but no such interference (10%) was observed for the direct HDL-C assay. The effect of serum triglyceride was determined by selecting 48 samples (triglyceride 1240 mg/L-28 600 mg/L) and comparing homogeneous HDL-C results with results of ultracentrifugation HDL-C (Fig. 1 ). Bias was calculated as follows: test method result minus ultracentrifugation method result. Ultracentrifugation of HDL-C was done at Wisconsin State Laboratory of Hygiene (Madison, WI). The 22 serum samples containing triglyceride concentrations <4000 mg/L (group 1) and the 20 serum samples containing triglyceride concentrations between 10 000 mg/L and 40 100 mg/L (group 2) were analyzed in a single run for HDL-C using these three homogeneous HDL-C reagents. Mean concentrations of HDL-C in group 1 were 508 mg/L for the direct HDL-C assay, 517 mg/L for the N-geneous^TM HDL-C assay, and 531 mg/L for the EZ-HDL^TM-C assay. However, mean concentrations of HDL-C in group 2 were 253 mg/L for the direct HDL-C assay, 352 mg/L for the N-geneous^TM HDL-C assay, and 253 mg/L for the EZ-HDL^TM-C assay. Marked differences were noted between assays in individual patients.

View larger version (15K): [in this window] [in a new window]   Figure 1. Plots of bias observed between reference method (ultracentrifugation) and each of three homogeneous HDL-C methods over a wide range of serum triglyceride concentrations. (Top) direct HDL-C; (middle) N-geneousTM HDL-C; and (bottom) EZ-HDLTM-C.

Sugiuchi et al. (6) and Harris et al. (12) reported precisions slightly better (CV <4%) than the results we obtained for direct HDL-C assay and N-geneous^TM HDL assay. This may be due to use of different analyzers or differences in the methods of calibration. The data in our study agree with Okamoto et al. (7), who reported that higher HDL-C concentrations were obtained by the direct method than by the precipitation method. Our finding of no statistically significant interference from bilirubin in any of the three homogeneous HDL-C assays agrees with the findings of Sugiuchi et al. (6) and Harris et al. (12). We found that hemoglobin produced a negative interference (>10%) with the N-geneous^TM HDL-C assay but had little effect on direct HDL-C assay and EZ-HDL^TM-C assay. Nauck et al. (5) reported that hemoglobin produced a positive interference (>10%) and bilirubin produced a negative interference (>10%) with a different homogeneous HDL-C assay, which uses PEG and antibodies against apo B and apo C-III (5). We found that there was a negative interference (>10%) by Intralipid^® at triglyceride concentrations >20 000 mg/L for the N-geneous^TM HDL-C and EZ-HDL^TM-C assays but not for the direct HDL-C assay. However, we found inconsistent HDL-C bias among high triglyceride concentration serum samples, suggesting that interference measurements using Intralipid may not truly represent the clinical situation. The negative bias of HDL-C concentration observed in serum of high triglyceride concentration (10 000 mg/L to 29 000 mg/L) using EZ-HDL^TM-C assay was much more apparent than with the other two homogeneous HDL-C assays. Harris et al. (12) reported a positive bias for N-geneous^TM assay with high triglyceride concentration (4000 mg/L to 10 000 mg/L) serum samples. We are unable to explain this discrepancy.

We conclude that these three homogeneous HDL-C assays are acceptable (total error <22%) for clinical laboratory use according to NCEP performance goals. These assays can shorten turnaround time and save labor costs. Overall, the direct HDL-C assay demonstrated slightly less interference from bilirubin, hemoglobin, and lipemia than the N-geneous^TM and EZ-HDL^TM-C assays. For accurate determination of HDL-C results in samples with lipemia and/or containing high concentrations of triglyceride, analysis by the ultracentrifugation reference method is indicated.

Acknowledgments

Boehringer Mannheim Corp. provided the direct HDL-cholesterol reagent kit and funded the ultracentrifugation HDL-C testing; Genzyme Diagnostics provided the N-geneous^TM HDL cholesterol reagent kit; and Sigma Diagnostics provided the EZ-HDL^TM cholesterol reagent kit. The Medical Editing Department, Kaiser Foundation Research Institute, provided editorial assistance.

Footnotes

fax 510-559-5204, e-mail Maggie.Lin{at}ncal.kaiperm.org

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