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Optimization of [beta]-quantification methods for high-throughput applications.

Cardiovascular disease (CVD)[1] continues to be the leading cause of death and morbidity for both men and women in the United States. The concentrations of various lipids and lipoproteins have been established as risk factors for CVD and serve as the basis for diagnosis of disease and also as indicators of therapy (1, 2). Improved methods for measurement of the concentrations of lipids and lipoproteins have evolved rapidly in the last decade to the point where concentrations of LDL- and HDL-cholesterol are now readily determined in a standard clinical laboratory without pretreatment of specimens to separate the lipoproteins. Despite these improvements, situations exist in which the use of traditional methods of lipoprotein measurement is desired.

[beta]-Quantification" is a generic term describing a variety of methods for measurement of lipoproteins following partial separation by ultracentrifugation. [beta]-Quantification is not standardized in terms of the volume of specimen analyzed, ultracentrifugation conditions, or analytical processes. However, when specific, well-defined steps are followed, [beta]-quantification is the basis for the reference methods for LDL- and HDL-cholesterol as practiced by the CDC (3). Despite the imperfect isolation of lipoproteins, such as the inclusion of intermediate density lipoproteins and lipoprotein(a) in the LDL fraction, [beta]-quantification has been used to establish the concentrations of the major classes of lipoproteins in most epidemiologic and clinical trials that have become the guidelines for risk assessment of CVD (1, 2, 4). Although not generally used to define CVD risk in the usual clinical setting, [beta]-quantification allows the measurement of VLDL lipid concentrations and also the concentrations of triglycerides in LDL and HDL, measurements that are sometimes required in clinical trials. Perhaps the most useful aspect of the [beta]-quantification method is the efficient removal of increased triglyceride-rich VLDL, which often interferes with the measurement of LDL- and HDL-cholesterol by other methods, particularly the estimation of LDL-cholesterol using the Friedewald equation, which loses validity for triglyceride concentrations >4.52 mmol/L (>400 mg/ dL) (5). Because the utility of the Friedewald equation is based on a defined ratio of triglycerides to cholesterol in VLDL, any treatment that alters that ratio of 5:1 (mass/ mass), such as treatment with estrogens, may provide inaccurate estimation of the LDL-cholesterol concentration. Finally, [beta]-quantification is useful for the clinical diagnosis of type III dysbetalipoproteinemia, wherein the ratio of VLDL-cholesterol to total triglyceride concentration is [greater than or equal to] 0.3 (6). In this disease, the concentration of LDL-cholesterol is significantly overestimated by the Friedewald equation, whereas [beta]-quantification provides the correct measurement.

Despite the necessity for [beta]-quantification in some clinical trials, drawbacks to its usefulness include the need for large specimen volumes, complex handling steps, and long ultracentrifugation times. The traditional [beta]-quantification method uses a 5-mL specimen, requiring the collection of relatively large amounts of blood, which may become prohibitive in clinical trials that involve children or frequent blood samplings over short time periods. The trend in approval of clinical trials by Institutional Review Boards is to minimize the amount of blood collected to reduce risk to the study participant. Furthermore, if repeat analysis is needed, it usually is not possible because of a lack of sufficient remaining specimen volume. The traditional [beta]-quantification method as practiced in the Lipid Research Clinics (LRC) Program also involves several technical steps that are inconvenient or involve a high degree of technical expertise, such as the use of time-consuming set-screw-sealed open-top tubes and a tube slicer, which requires substantial force and extensive experience for successful use. Such steps do not adapt well to high-throughput analysis. Finally, the standard 18-h ultracentrifugation run time precludes rapid turnaround time, which is sometimes required in clinical trials, particularly in the prerandomization phase of a trial, by adding a full day to the time of analysis, for a total of 2.5 days. To minimize these deficiencies, we have adapted the traditional [beta]-quantification method for hight-hroughput and rapid turnaround time by developing two modified [beta]-quantification methods. Other low-volume, high-speed [beta]-quantification methods using tabletop ultracentrifuges have been described; however, large numbers of specimens cannot be processed in these systems (7). Using the modifications described herein, we routinely analyze up to 80 specimens per day in this laboratory.

Materials and Methods

SPECIMEN COLLECTION

Fresh human whole blood was collected by venipuncture in glass SST Vacutainer Tubes lacking an anticoagulant and allowed to clot at room temperature for 30 min. Serum was separated by low-speed centrifugation for 7500 g X min. For the intrarun precision study, a pool of fresh serum was prepared by combining residual clinical specimens for evaluation of the 5- and 1-mL methods. A fresh serum specimen from a single donor was used to evaluate the 0.23-mL method.

ULTRACENTRIFUGATION

All ultracentrifugation equipment and supplies were from Beckman Instruments. The equipment and conditions used for each method are summarized in Figs. 1-3 and Table 1. The 5-mL method followed the ultracentrifugation methods of the LRC Program for the analysis of lipoproteins (8) with two modifications: heat-sealed ultracentrifugation tubes were used (9), and cholesterol concentration was measured by a CDC-standardized enzymatic assay rather than by the Abell-Kendall reference method because the 0.23-mL method did not provide sufficient specimen for reference method analysis. In addition, because the LRC method requires the measurement of HDL-cholesterol concentration on the nonultracentrifuged serum specimen rather than the bottom fraction, a single HDL-cholesterol measurement could be made on serum that would serve as a constant factor for the calculation of LDL-cholesterol concentration for each method: LD[L.sub.5, 1, or 0.23] mL = Bottom [fraction.sub.5, 1, or 0.23] [mL.sup.-] [HDL.sub.all]. In this regard, the variability of the HDL measurement would not affect the evaluation of the ultracentrifugation separation step, which then becomes of matter of a single measurement of cholesterol concentration in the bottom fraction.

Precooled rotors were ultracentrifuged in either a Beckman L7-55 or a L8-55 ultracentrifuge that had been precooled to 10[degrees]C. Specimens were added to the appropriate ultracentrifuge tube at native density, using a Class A volumetric glass pipette for the 5-mL specimen, a Rainin EDP2 1000-[micro]L pipette for the 1-mL specimen, and a Rainin EDP2 250-[micro]L pipette for the 0.23-mL specimen. The 5-mL specimens were overlaid with sufficient EDTA-saline buffer (1 mmol/L EDTA, 9 g/L NaCl, pH 8.2) to completely fill the neck of the centrifuge tube before sealing with heat (9). Rotors were ultracentrifuged at 10[degrees]C for the indicated time (Table 1). For the Type 25 rotor, only the outermost row was used in this study, providing space for 44 tubes. The middle and innermost rows could be used, but the extended ultracentrifugation times required for complete separation of top and bottom fractions as a result of the shorter radii precluded their use in high-throughput laboratory situations. To avoid mixing of separated lipoproteins, rotors were stopped using the "Slow Acceleration" feature of the ultracentrifuge, in which the rotor slowly comes to speed initially followed by full dynamic braking at the end of the run until 800 rpm was attained, after which the rotor was allowed to coast unimpeded until it stopped.

[FIGURE 1 OMITTED]

POSTULTRACENTRIFUGATION PREPARATION OF 5-mL SPECIMEN

[beta]-Quantification depends on the physical separation of VLDL from other lipoproteins by ultracentrifugation, collection of the fractions, and finally reconstitution of the fractions back to the original specimen volume. After ultracentrifugation, the top and bottom fractions were separated in the ultracentrifugation tube with a blade, using a tube slicer (Nuclear Supply Company), by cutting in the clear zone between the top and bottom fractions. This process required the technician to hold the tube in place in the slicer with one hand and then abruptly strike the blade of the slicer with the palm of the other hand with sufficient speed and force to drive the blade through the tube without remixing the contents. The top fraction, which contained VLDL, was transferred to a 5.0-mL volumetric flask, using a disposable glass transfer pipette. The top of the tube was washed with EDTA-saline buffer, which was added to the volumetric flask. After all of the top fraction was transferred to the volumetric flask, the slicer blade was drawn back and the contents of the bottom fraction were transferred to another 5.0-mL volumetric flask. Resuspension of the congealed pellet of serum proteins in the bottom of the tube required extensive manipulation with the tip of the pipette. Care was taken to quantitatively transfer the contents of both fractions of the sliced centrifuge tube to the appropriate volumetric flask. After storage overnight in a refrigerator to allow the dissipation of bubbles, each fraction was brought to volume (5.0 mL) with EDTA-saline after the contents were warmed to ambient temperature and visual inspection confirmed that all of the pellet had dissolved. When processed properly, the concentration of lipoprotein in each fraction would then be the same as in the nonfractionated serum.

[FIGURE 2 OMITTED]

POSTULTRACENTRIFUGATION PREPARATION OF 1- AND 0.23-mL SPECIMENS

After ultracentrifugation, the top and bottom fractions were separated using a Beckman CentriTube Slicer (Fig. 3). Unlike the slicer used for 5-mL tubes, the CentriTube Slicer required only finger-tip pressure on the "Blade Advance Knob" to smoothly pull the "Adapter Plate" and "Blade" through the thick-walled tubes. This form of slicing was less disruptive to the separated fractions than was the process used for the 5-mL method. The proper position of the cut was determined empirically by pipetting either 1 or 0.23 mL of saline, as appropriate, into the correct-sized ultracentrifuge tube, slicing the tube, and then measuring the volume of saline in the top fraction using gravimetrically calibrated pipettes. To fine tune the position of the cut, the "Tube Elevation Knob" was adjusted until the proper volume of saline was collected from the top fraction: 400 [micro]L for the 1-mL method and 70 [micro]L for the 0.23-mL method. For convenience of use, the current calibrated positions of the "Index Bar" and the Tube Elevation Knob were marked on tape applied to the slicer. Calibration of the CentriTube Slicer was verified monthly, with recalibration as necessary.

After slicing, the top fractions were transferred to either 12 X 75 mm glass culture tubes (1-mL method) or analyzer cups (0.23-mL method), using calibrated P-200 pipetters. Residual lipoproteins in the top fraction were collected by washing the cut centrifuge tube with either 600 [micro]L (1-mL method) or 160 [micro]L (0.23-mL method) of EDTA-saline in two steps (see Fig. 1 and Table 1) and transferring the wash to the appropriate tube or analyzer cup. After the blade was drawn back, the bottom fractions were transferred to clean 12 X 75 mm glass culture tubes (1-mL method) or analyzer cups (0.23-mL method), using calibrated P-200 pipetters. Residual lipoproteins in the bottom fraction were collected by washing the cut centrifuge tube with either 400 [micro]L (1-mL method) or 70 [micro]L (0.23-mL method) of EDTA-saline and transferring the wash to the appropriate tube or analyzer cup. Protein pellets in the bottom fraction were readily redissolved by gentle mixing using the pipette tip. By the combination of these washes, each fraction was diluted to the original specimen volume (1 or 0.23 mL). The reconstituted specimens were mixed with a vortex-type mixer for 20 s before being analyzed for cholesterol and triglyceride concentrations.

[FIGURE 3 OMITTED]

Because of the importance of delivery of accurate volumes of specimen to the ultracentrifugation tube and of wash solutions during the reconstitution steps for the land 0.23-mL methods, the accuracy of the process was assessed gravimetrically. Water was pipetted into 10 tared ultracentrifugation tubes for each method, using the appropriate calibrated pipette; the ultracentrifugation tubes were then reweighed. The mean accuracy ([+ or -] CV) was 99.3% ([+ or -] 0.42%) for the 1-mL method and 98.9% (0.75%) for the 0.23-mL method. The tubes were then sliced in calibrated slicers, processed as usual using calibrated pipettes, and the recoveries of the reconstituted top and bottom fractions were determined gravimetrically. The mean recoveries for the bottom fractions were 99.4% ([+ or -] 0.45%) and 98.8% ([+ or -] 1.32%) for the 1- and 0.23-mL methods, respectively, and for the top fractions were 97.4% ([+ or -] 0.77%) and 90.0% ([+ or -] 1.75%) for the 1- and 0.23-mL methods, respectively.

CHOLESTEROL AND TRIGLYCERIDE ANALYSIS

Cholesterol concentrations were determined enzymatically (cholesterol esterase/oxidase/peroxidase) using Technicon RA[R] reagent sets (Bayer Corp.) on a Technicon RA-1000 Analyzer (Miles, Inc.). Triglyceride concentrations also were measured enzymatically (lipase/kinase/ oxidase/peroxidase) using Technicon RA reagent sets on a Technicon RA-1000 Analyzer. All triglyceride measurements were corrected for the presence of endogenous free glycerol in the serum sample by subtracting glycerol blank values. Lipid methods were standardized under the CDC-National Heart, Lung, Blood Institute Lipid Standardization Program and had intra- and interrun imprecision of <2% (3). All analyses were performed in accordance with the manufacturers' recommendations.

RECOVERY QUALITY CONTROL

The validity of the reconstitution and analytical steps for all three [beta]-quantification methods was verified by checking individual sample recovery: Top cholesterol + Bottom cholesterol = Total cholesterol within 93% and 103%. Similarly, triglyceride summation recovery verification criteria were within 90% and 110%. In routine use, specimens that fell outside these ranges would have been reanalyzed for cholesterol and triglyceride concentrations to verify the analytical measurement. If the repeat measurements confirmed the original values and the recoveries were still out of the stated limits, suggesting the possibility of a reconstitution error, the specimen would be subjected to repeat ultracentrifugation.

FRESH PATIENT SPECIMEN METHOD COMPARISON

Forty-five fresh human whole blood samples were collected and processed to yield fresh serum. Each sample was analyzed by the 5-, 1-, and 0.23-mL [beta]-quantification methods. The ultracentrifugations were timed such that cholesterol and triglyceride concentrations were measured in the top and bottom fractions in the same analytical run to eliminate the effects of interrun analytical variability, so that any observed differences between methods would be attributable solely to differences in the ultracentrifugation and processing steps.

INTRARUN PRECISION AND DETERMINATION OF ULTRACENTRIFUGATION ZONAL STABILITY

In high-throughput situations, many specimens must be processed simultaneously; thus, some specimens stand longer times after ultracentrifugation before being sliced than others, allowing time for remixing of the separated fractions by diffusion. To evaluate the stability of ultracentrifuged samples, 17 replicates were analyzed by the 5-, 1-, and 0.23-mL [beta]-quantification methods. After ultracentrifugation, tubes were sliced at 15-min intervals over a period of 4 h. Cholesterol and triglyceride concentrations were determined in the top and bottom fractions.

COMPARISON OF 1-mL METHOD TO REFERENCE CDC [beta]-QUANTIFICATION METHOD

The 1-mL method is used most commonly in this laboratory for clinical trials. To evaluate the precision and accuracy of this method relative to the CDC reference methods for LDL and HDL, a comparison was made through the CDC's Cholesterol Reference Method Laboratory Network program. The CDC provided three challenges over 16 months. Each challenge involved four frozen serum pools, each of which was subjected to [beta]-quantification by the 1-mL method in duplicate at weekly intervals for 4 weeks; sufficient material was not available to evaluate the 5- and 0.23-mL methods. The cholesterol concentration was measured in duplicate in the bottom fraction and in the HDL supernate prepared from the bottom fraction (to meet the requirements of the CDC comparison design) by the dextran sulfate [MT 50 000 method of Warnick et al. (10)]. LDL-cholesterol was calculated by subtracting the HDL-cholesterol concentration from the bottom fraction cholesterol concentration. Therefore, for each challenge, each of four pools was subjected to [beta]-quantification 8 times, and the cholesterol concentrations were measured 16 times. Data from this comparison were used to evaluate interrun precision for the 1-mL method.

STATISTICS

The differences in mass, percent bias, and relationship between bottom fraction cholesterol concentration for each modified method relative to the 5-mL method were determined by paired Student's Mest, 95% confidence intervals, and linear regression, respectively. Statistical analysis was performed using the Sigma Stat program (SPSS, Inc.).

Results COMPARISON of 1- AND 0.23-mL METHODS TO 5-mL

METHOD

The 5-mL [beta]-quantification method used in the LRC Program (8) is used as the in-house reference method for the Core Laboratory for Clinical Studies. Because the LDL-cholesterol concentration is determined by subtracting the HDL-cholesterol concentration, derived from the nonultracentrifuged serum, from the cholesterol concentration in the bottom fraction, the ultracentrifugation conditions for the modified methods were optimized to provide cholesterol concentrations in the bottom fraction equivalent to that of the 5-mL method. Thus, equivalent bottom fraction cholesterol concentrations would yield equivalent LDL-cholesterol concentrations because a common HDL-cholesterol value would be used among the methods. Forty-five fresh human specimens with total cholesterol of 139-389 mg/dL and triglycerides of 66-875 mg/dL were analyzed by the three methods. Mean bottom fraction cholesterol concentrations (mg/ dL) were 175.5, 177.1, and 177.9 for the 5-,1-, and 0.23-mL methods, respectively (Table 2). Although paired Mest analysis showed that both modified methods were significantly different from the 5-mL method, the differences were clinically insignificant. On average, the bottom fraction cholesterol concentrations (mean [+ or -] SD) were slightly higher than the 5-mL method by 0.8% ([+ or -] 3.4%) and 1.5% ([+ or -] 2.9%) for the 1- and 0.23-mL methods, respectively. The 95% confidence interval for the percent bias relative to the 5-mL method contained zero bias for the 1-mL method (Table 2), whereas the lower interval for the 0.23 method began at 0.6%. Linear regression analysis provided the following equations for bottom fraction cholesterol concentration: 1 mL = (1.047 X 5 mL) - 6.5 (r = 0.9986) and 0.23 mL = (1.030 X 5 mL) - 2.6 (r = 0.9986).

This comparison also provided an estimate of the recoveries of cholesterol and triglycerides in each fraction (Table 2), which is used in this laboratory as a quality-control check of the reconstitution and analytical steps for each individual specimen. Overall, all three methods had mean recoveries of 97.1-104.2%, with the 1-mL method closest to 100% recovery for both lipids. Both the 5- and 1-mL methods had three failures each of internal quality control, attributable to unacceptable recovery of either cholesterol or triglycerides (6.7% failure rate). In routine use, specimens failing the internal quality control would have been repeated. The 0.23-mL method had a higher apparent failure rate of 20% (9 of 45). However, 3 specimens failed for both cholesterol and triglycerides, which would have required the repeat of 6 of the specimens rather than 9, for an effective failure rate of 13.3% (6 of 45).

INTRARUN PRECISION AND DETERMINATION OF ULTRACENTRIFUGATION ZONAL STABILITY

To evaluate the intrarun precision as well as the extent of remixing of the top and bottom fractions on standing after ultracentrifugation, 17 replicates were analyzed in a single ultracentrifugation run by each of the three [beta]-quantification methods. After ultracentrifugation, tubes were sliced and processed at 15-min intervals over a 4-h period (Fig. 4). The intrarun imprecision of the 5- and 1-mL methods was equivalent for cholesterol in the bottom fraction, with CVs of 0.96%, whereas the CV for the 0.23-mL method was 1.7%. No remixing of top and bottom fractions that would affect the concentration of cholesterol in the bottom fraction was observed for any of the methods for up to 4 h, indicating that during that time period a technician may process as many as 48 specimens at a rate of 5 min/specimen. In addition, none of the individual analyses for the 5- or 1-mL methods failed the recovery quality-control check; however, three of the time points for the 0.23-mL method failed. Given the distribution of failures (cholesterol at 0 and 225 min and triglycerides at 210 min), the failures appeared to be attributable to sporadic imprecision, rather than a trend.

[FIGURE 4 OMITTED]

COMPARISON OF 1-mL METHOD TO REFERENCE CDC [beta]-QUANTIFICATION METHOD

The separation of cholesterol into the bottom fraction was shown to be equivalent among the three methods. The next step was to evaluate the accuracy of the measurement of the LDL-cholesterol concentration. Although the National Cholesterol Education Program (NCEP) has set performance goals for the accuracy of the measurement of LDL- and HDL-cholesterol concentrations, no programs exist that allow clinical laboratories to make comparisons to the reference methods for these analytes. By virtue of its participation in the Cholesterol Reference Method Laboratory Network, the Core Laboratory for Clinical Studies was able to perform such a comparison with the CDC for the 1-mL [beta]-quantification method. Both LDL-and HDL-cholesterol concentrations met the NCEP goal for accuracy (HDL bias [less than or equal to] 5% and LDL bias [less than or equal to] 4%; measured mean bias, -2.81% and 2.19%, respectively) and imprecision (HDL CV [less than or equal to] 4% and LDL CV [less than or equal to] 4%; measured mean imprecision, 1.8% and 1.6%, respectively). NCEP goals for total error [% bias + 2(% CV)] for HDL and LDL are 13% and 12%, respectively. The 1-mL method had total errors of 6.45% and 5.43%, respectively. Although the NCEP has no goals for accuracy, precision, or total error for the measurement of the cholesterol concentration in the bottom fraction, the 1-mL [beta]-quantification method had a bias of 0.83%, a CV of 1.4%, and a total error of 3.69% vs the CDC reference method. In comparison, the typical performance of the LDL-cholesterol reference method at the CDC is at a CV of 1.0-1.3% for LDL-cholesterol and 0.8-1.1% for the bottom fraction cholesterol (3).

Discussion

Lipoproteins are a heterogeneous population of macromolecules that, among other diverse functions, transport lipids in the water phase of blood. Certain classes of lipoproteins, such as LDL, interact with vascular tissue and deposit lipids in the vascular wall, which leads to plaque formation and atherosclerosis. Conversely, other classes of lipoproteins, such as HDL, remove excess cholesterol from vascular tissue for eventual removal from the body. Measurement of the relative amounts of these and other lipoproteins has been used to assess the risk of CVD. Until recently, the determination of lipoprotein classes was based on the physical separation of lipoproteins followed by the measurement of cholesterol, with the concentration of cholesterol serving as a surrogate indicator of the amount of lipoprotein in the circulation. Technology has advanced to the point where both LDL- and HDL-cholesterol concentrations can be measured in intact serum without physical separation of the various lipoproteins. Although this progress has improved the measurement of LDL and HDL for the purpose of CVD risk assessment, the concentrations of other important lipoproteins are not readily determined by similar methods, particularly in research settings. In addition, subtle changes in the composition of lipoproteins, such as the ratio of lipid to protein in LDL, which determines particle size, cannot be obtained by the current homogeneous methodologies. Similarly, pharmacologic or environmental perturbations that alter the composition of lipoproteins, particularly VLDL, may lead to errors in the measurement of LDL-cholesterol when calculated by the Friedewald equation. The partial physical separation of lipoproteins by [beta]-quantification also allows the measurement of both cholesterol and triglycerides in VLDL, LDL, and HDL as well as the apolipoprotein B concentration in LDL. Finally, specimens that are excessively hypertriglyceridemic are problematic for the measurement of LDL and HDL by both the homogeneous and traditional methods unless the triglyceride-rich VLDL is removed by ultracentrifugation. Therefore, the [beta]-quantification method is still relevant despite the arrival of new homogeneous assays for LDL- and HDL-cholesterol, particularly in basic and clinical research, where rapid turnaround time and small specimen size often are critical.

Despite these needs and the previous publication of modifications to the traditional 5-mL [beta]-quantification method, well-characterized modifications that allow the processing of large numbers of specimens using small specimen volumes and short turnaround times are lacking. We describe here two lower-volume, high-throughput methods that fulfill these needs, agree with the traditional method in terms of precision and accuracy, and for the 1-mL method, meet the requirements of the NCEP for the measurement of LDL- and HDL-cholesterol concentration.

Despite the finding of statistically significant differences by paired t-test between the bottom fraction cholesterol concentration of the modified methods and the 5-mL method, the differences were small, 1.8 mg/dL (0.8%) and 2.6 mg/dL (1.5%) for the 1- and 0.23-mL methods, respectively. Such differences are not significant clinically and are well within the similar NCEP guidelines for acceptable bias for LDL-cholesterol, which is <4% bias. True bias for the LDL-cholesterol concentration, rather than for the bottom fraction cholesterol concentration compared with our in-house 5-mL method, was assessed by comparison to the CDC reference method. By this comparison, the 1-mL method met the NCEP guidelines for bias, precision, and total error.

For a traditional lipid-specialty laboratory, the processing of large numbers of specimens (>18) presents challenges that can be overcome by the use of an efficient laboratory system and several technicians well trained in the [beta]-quantification method. We have incorporated several steps into the described modifications that allow convenient specimen handling while maintaining high accuracy and precision, which may otherwise be lost with the use of smaller specimen volumes. Factors that increased specimen throughput were (a) use of a gravimetrically calibrated manual pipette for the introduction of specimen into the ultracentrifuge tube; (b) use of open-top ultracentrifuge tubes that do not require sealing before ultracentrifugation; (c) use of a calibrated CentriTube Slicer; (d) use of a set of dedicated, preset, and gravimetrically calibrated manual pipettes for the reconstitution of the top and bottom fractions; and (e) placement of reconstituted fractions directly into analyzer-ready tubes or cups rather than volumetric flasks. The overall time savings were substantial: the 1- and 0.23-mL methods required only 20 and 6 h, respectively, compared with 2.5 days for the 5-mL method. As a beneficial consequence of the ultracentrifugation conditions, the protein pellet in the bottom of the centrifuge tube is less compact than that of the 5-mL method and readily resolublizes during the reconstitution and washing steps, saving considerable time. In addition, the top and bottom fractions do not remix for at least 4 h after ultracentrifugation, allowing a single experienced technician to process a large number of specimens.

To maintain the accuracy and precision of the methods, a set of three dedicated pipettes are devoted to each workstation. The pipettes are regularly calibrated gravimetrically and are preset to the volume required for each pipetting step. Therefore, the time-consuming and tedious process of resetting each volume during the reconstitution step, as well as the possibility for errors or increasing imprecision attributable to pipette backlash, are avoided. Similarly, the CentriTube Slicer is calibrated regularly to provide consistency in the position of the tube slice. Because reconstitution of the fractions involves the addition of a calculated volume of diluent rather than the use of a volumetric flask, if the slice is not at the correct position, the volumes of the reconstituted fractions as well as the measured concentrations of cholesterol and triglycerides will be in error. For this reason, the recoveries of both cholesterol and triglycerides are calculated for each specimen. The limits for the recovery quality control were set based on what would be considered desirable performance.

The limits may appear wider than may be expected but are reasonable because of at least three factors: (a) the final evaluation is based on the combination of three measurements, each with its own analytical variability that contributes to the overall variability of the process; (b) "failure" can be attributable to either cholesterol or triglycerides or to both lipids, which substantially increases the likelihood of failure; and (c) limits are extended on the low side of recovery because of the known problems with quantitative recovery of the top fraction. In addition, the limits approximate the NCEP precision recommendations for cholesterol (2 SD = [+ or -] 6%) and triglyceride (2 SD = [+ or -] 10%) measurements. In general, the use of recovery quality control is not commonly practiced. Informal surveying has shown that of eight laboratories routinely performing [beta]-quantification methods, four have no form of recovery quality control whatsoever, three perform recovery quality control for either cholesterol or triglycerides and then only on a limited number of specimens, and one performs it for both lipids on every specimen analyzed.

Each modified [beta]-quantification method has advantages depending on the purpose for which the assay is being run. All three methods allowed at least 4 h of standing time without significant remixing of fractions. The 0.23-mL method provided rapid turnaround time because of its 4-h ultracentrifugation time; repeats could be conducted quickly if necessary and required only -5% of the volume of the 5-mL method. In our laboratory, apolipoproteins A-I and B sometimes are analyzed in the top and bottom fractions from the 0.23-mL method, in addition to cholesterol and triglycerides. However, the small specimen volume precludes the measurement of HDL-cholesterol in the bottom fraction of hypertriglyceridemic specimens by precipitation methods unless replicate specimens are run and pooled.

The 0.23-mL method appeared to be the most variable of the three methods compared in that it had the highest failure rate, 13.3% vs 6.7% for the 5- and 1-mL methods. This variability was also observed in the intrarun precision study in which the 0.23-mL method had the largest CV for either lipid in both the top and bottom fractions. Combining these less precise values, obviously, led to a higher failure rate for the 0.23-mL method. The most variable measurement was for the concentration of triglycerides in the bottom fraction, with CVs of 2.0%, 4.5%, and 13% for the 5-,1-, and 0.23-mL methods, respectively. In addition, the concentration of triglycerides in the bottom fraction of the 0.23-mL method, which had the shortest separation pathway length of the three methods, increased slightly over time (Fig. 4), suggesting a possible remixing of top and bottom fractions upon standing. However, the top fraction triglyceride concentration did not appear to decrease, and the bottom fraction cholesterol did not increase.

Appropriate technical expertise is essential for the success of this method because of the handling of many small volumes; however, as technical experience is gained the failure rate may approach that of the other methods. As might be expected, the 1-mL method is intermediate between the 5- and 0.23-mL methods in terms of convenience and practical usefulness. Technical experience is somewhat less critical relative to the 0.23-mL method, and variability is comparable to or less than that of the 5-mL method. The 1-mL method also allows HDL-cholesterol to be measured directly in the bottom fraction by precipitation methods, as well as the analysis of cholesterol by the Abell-Kendall reference method, which requires at least 0.25-mL of specimen for analysis. As many as 80 specimens per day are routinely processed in a 2-h period by the 1-mL method using two rotors and two ultracentrifuges and the services of four technicians for simultaneous slicing of the tubes. Therefore, based on our experience, the 1-mL method provides the necessary reliability and throughput required by large-volume, high-quality clinical research trials.

References

(1.) Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. The Expert Panel. Arch Intern Med 1988; 148:36-69.

(2.) Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA 1993;269:3015-23.

(3.) Myers GL, Cooper GR, Greenberg N, Kimberly MM, Waymack PP, Hassemer DJ. Standardization of lipid and lipoprotein measurements. In: Rifai N, Warnick GR, Dominiczak MH, eds. Handbook of lipoprotein testing, 2nd ed. Washington, DC: AACC Press, 2000: 717-48.

(4.) The Lipid Research Clinics Coronary Primary Prevention Trial results. I. Reduction in incidence of coronary heart disease. JAMA 1984;251:351-64.

(5.) Friedewald WT, Levy RT, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without the use of the preparative ultracentrifuge. Clin Chem 1972;18: 499-502.

(6.) Mahley RW, Rail SC Jr. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 7th ed. New York: McGraw-Hill, 1995:1953-80.

(7.) Cathcart S, Dominiczak MH. The measurement of lipoprotein subfractions in plasma using a tabletop ultracentrifuge. Ann Clin Biochem 1990;27:459-64.

(8.) Manual of laboratory operations for the Lipid Research Clinics Program. In: Hainline A, Karon J, Lippel K, eds. Lipid and lipoprotein analysis, 2nd ed. Bethesda MD: National Heart, Lung, Blood Institute, National Institutes of Health, 1982:63-77.

(9.) Cole TG, Gibson DW. Evaluation of heat-sealed ultracentrifuge tubes in the isolation of plasma lipoproteins. J Lipid Res 1984; 25:312-4.

(10.) Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982;28:1379-88.

THOMAS G. COLE, * CONSTANCE A. FERGUSON, DAVID W. GIBSON, and WILLIAM L. NOWATZKE

Core Laboratory for Clinical Studies, Washington University School of Medicine, St. Louis, MO 63110. This work appeared previously in abstract form (Clip Chem 2000;46:A95).

[1] Nonstandard abbreviations: CVD, cardiovascular disease; LRC, Lipid Research Clinics; and NCEP, National Cholesterol Education Program.

* Address correspondence to this author at: Core Laboratory for Clinical Studies, Washington University School of Medicine, Box 8046, 660 S. Euclid Ave., St. Louis, MO 63110. Fax 314-362-4782; e-mail Thom@im.wustl.edu.

Received October 3, 2000; accepted January 30, 2001.
Table 1. Ultracentrifugation equipment and conditions.

 Method

 5-mL 1-mL
Ultracentrifugation

Rotor Type 50.3 Ti Type 25
Tube Polyallomer bell top Polycarbonate
 quick seal (cat. (cat. no.
 no. 344619) 355657)
Time, h 18 18
Speed, rpm 40000 25000
Temperature, [degrees]C 10 10
Average radius, mm 64.20 122.80
g X h 2070828 1546650

Slicing

From bottom, cm 2.70 NA'
Vertical scale, cm NA -y1.9
Tube elevation knob, cm NA -1.25
Top fraction, [micro]L NA 400
Bottom fraction, [micro]L NA 600

Top transfer/wash

Transfer top to: 5-mL volumetric 12 X 75 mm
 flask culture tube
Wash, (b) [micro]L ~750 300
Wash, [micro]L ~750 300

Resuspend pellet

Transfer bottom to: 5-mL volumetric 12 X 75 mm
 culture tube
Wash, [micro]L ~750 400
Wash, [micro]L ~750
 Bring to volume Analyze directly
 and analyze
Total time to analvsis 2.5 davs 20 h

 Method

 0.23-mL
Ultracentrifugation

Rotor Type 42.2 Ti
Tube Cellulose propionate
 (cat. no. 342303)

Time, h 4
Speed, rpm 40000
Temperature, [degrees]C 10
Average radius, mm 108.50
g X h 777728

Slicing

From bottom, cm NA
Vertical scale, cm -y0.6
Tube elevation knob, cm -1.35
Top fraction, [micro]L 70
Bottom fraction, [micro]L 160

Top transfer/wash

Transfer top to: Sample cup

Wash, (b) [micro]L 70
Wash, [micro]L 90

Resuspend pellet

Transfer bottom to: Sample cup

Wash, [micro]L 70
Wash, [micro]L
 Analyze directly

Total time to analvsis 6 h

(a) NA, not applicable.

(b) Wash = 1 mmol/L EDTA, 9 g/L NaCl, pH 8.2.

Table 2. Fresh specimen method comparison. (a)

 Bottom fraction cholesterol

Method mg/dL ([+ or -] SD) Bias vs 5-mL, % (95% Cl) (b)

5-mL 175.3 ([+ or -] 44.0)
1-m L 177.1 ([+ or -] 46.4) 0.8 (-0.2 to 1.9)
 P = 0.046
0.23-mL 177.9 ([+ or -] 45.6) 1.5 (0.6 to 2.4)
 P = 0.0009

 Recovery, %, mean ([+ or -] SD), [number of failures]

Method Cholesterol Triglycerides

5-mL 97.5 ([+ or -] 2.1) [2] 97.1 ([+ or -] 2.7) [1]
1-m L 100.0 ([+ or -] 2.6) [3] 99.4 ([+ or -] 2.6) [0]

0.23-mL 101.0 ([+ or -] 2.3) [6] 104.2 ([+ or -] 4.5) [3]

(a) Fresh serum specimens (n = 45) were analyzed by each of the
methods. Failures indicate the number of analyses for which the
sum of top and bottom fraction lipid concentrations did not meet
the acceptance criteria for internal recovery quality control.
Paired t-test was used to establish statistical significance vs
the 5-mL method for the bottom fraction cholesterol concentration.

(b) CI, confidence interval.
COPYRIGHT 2001 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2001 Gale, Cengage Learning. All rights reserved.

Article Details
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Title Annotation:Lipids, Lipoproteins, and Cardiovascular Risk Factors
Author:Cole, Thomas G.; Ferguson, Constance A.; Gibson, David W.; Nowatzke, William L.
Publication:Clinical Chemistry
Article Type:Report
Date:Apr 1, 2001
Words:6065
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