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1
Core Laboratory for Clinical Studies, Washington University School of Medicine, St. Louis, MO 63110.
a 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{at}im.wustl.edu.
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Abstract |
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Methods: Two lower-volume modifications of the traditional 5-mL ß-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 ß-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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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"ß-Quantification" is a generic term describing a variety of
methods for measurement of lipoproteins following partial separation by
ultracentrifugation. ß-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, ß-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, ß-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, ß-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 ß-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, ß-quantification is useful
for the clinical diagnosis of type III dysbetalipoproteinemia, wherein
the ratio of VLDL-cholesterol to total triglyceride concentration is
0.3 (6). In this disease, the concentration of
LDL-cholesterol is significantly overestimated by the Friedewald
equation, whereas ß-quantification provides the correct measurement.
Despite the necessity for ß-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 ß-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 ß-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 ß-quantification method for high-throughput and rapid turnaround time by developing two modified ß-quantification methods. Other low-volume, high-speed ß-quantification methods using table-top 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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: LDL5, 1, or 0.23
mL = Bottom fraction5, 1, or 0.23 mL
- HDLall. 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.
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Precooled rotors were ultracentrifuged in either a Beckman L7-55 or a
L8-55 ultracentrifuge that had been precooled to 10 °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-µL pipette for the 1-mL specimen, and a Rainin EDP2
250-µL 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 °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.
postultracentrifugation preparation of 5-mL specimen
ß-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.
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
µL for the 1-mL method and 70 µL 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 µL (1-mL method) or 160 µL (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 µL (1-mL method) or 70 µL (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.
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 1- and 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 (± CV) was 99.3% (± 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% (± 0.45%) and 98.8% (± 1.32%) for the 1- and 0.23-mL methods, respectively, and for the top fractions were 97.4% (± 0.77%) and 90.0% (± 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® 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 ß-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 ß-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 ß-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 ß-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 ß-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
[Mr 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 ß-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
t-test, 95% confidence intervals, and linear regression,
respectively. Statistical analysis was performed using the Sigma Stat
program (SPSS, Inc.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although paired t-test 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 ± SD) were slightly
higher than the 5-mL method by 0.8% (± 3.4%) and 1.5% (± 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).
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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 ß-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.
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comparison of 1-mL method to reference cdc ß-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
ß-quantification method. Both LDL- and HDL-cholesterol concentrations
met the NCEP goal for accuracy (HDL bias 
5% and LDL bias 4%;
measured mean bias, -2.81% and 2.19%, respectively) and imprecision
(HDL CV 4% and LDL CV 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
ß-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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite these needs and the previous publication of modifications to the traditional 5-mL ß-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 ß-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 = ± 6%) and triglyceride (2 SD = ± 10%) measurements. In general, the use of recovery quality control is not commonly practiced. Informal surveying has shown that of eight laboratories routinely performing ß-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 ß-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.
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Footnotes |
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1 Nonstandard abbreviations: CVD, cardiovascular disease; LRC, Lipid Research Clinics; and NCEP, National Cholesterol Education Program. 
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The following articles in journals at HighWire Press have cited this article:
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  M. Cuchel, L. T. Bloedon, P. O. Szapary, D. M. Kolansky, M. L. Wolfe, A. Sarkis, J. S. Millar, K. Ikewaki, E. S. Siegelman, R. E. Gregg, et al. Inhibition of Microsomal Triglyceride Transfer Protein in Familial Hypercholesterolemia N. Engl. J. Med., January 11, 2007; 356(2): 148 - 156. [Abstract] [Full Text] [PDF]
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 M. Nauck, G. R. Warnick, and N. Rifai Methods for Measurement of LDL-Cholesterol: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation Clin. Chem., February 1, 2002; 48(2): 236 - 254. [Abstract] [Full Text] [PDF]
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, bottom fraction triglycerides; 
, top fraction
cholesterol; 
, bottom fraction cholesterol.