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Articles |
1
University of Freiburg, Freiburg, Germany.
2
Evaluation Department, Boehringer Mannheim GmbH,
Mannheim, Germany.
3
Academic Hospital Rotterdam, Rotterdam, The Netherlands.
4
University of Münster, Münster, Germany.
5
University of Gent, Gent, Belgium.
6
Kreiskrankenhaus Bruchsal, Bruchsal, Germany
7
Gemeinschaftspraxis Heidelberg, Heidelberg, Germany.
a Address correspondence to this author at: Department of Medicine, Division of Clinical Chemistry, University Hospital of Freiburg, Hugstetter Str. 55, 79106 Freiburg i. Br., Germany. Fax +49-761-270 3444.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Together with total cholesterol and triglycerides, the determination of HDL-C allows the calculation of LDL-C according to Friedewald et al. (10).
For these reasons, reliable and easy-to-perform methods are needed to quantify HDL-C.
Measurement of HDL-C is still critical from the view of the clinical chemist, because substantial deviations between different laboratories have been observed and because the conventional precipitation procedures are time consuming (11)(12)(13)(14). In addition, they do not meet the HDL-C performance goals of the CDC for 1998, which require for HDL-C a CV of <4% at concentrations >420 mg/L and a SD <17 mg/L at concentrations <420 mg/L (15).
Most frequently, HDL-C is measured in the supernatant after precipitation of apolipoprotein (apo) B-containing lipoproteins by dextran sulfate or phosphotungstic acid (PTA)/MgCl2 (11)(12)(13)(14). All of these methods involve both a precipitation and a centrifugation step, which prevent full automation. In recent years the costs of laboratory procedures have attracted increasing attention. Methods that eliminate manual handling, such as pretreatment with a precipitation reagent in the case of the HDL-C determination, may considerably reduce costs.
Previously, new procedures for the determination of HDL-C were published. They have in common the avoidance of at least the tedious centrifugation step (16)(17)(18).
This multicenter study was performed in seven laboratories from three
European countries. We evaluated a homogeneous HDL-C assay that
includes polyethylene glycol (PEG)-modified enzymes and
-cyclodextrin sulfate to selectively determine HDL-C in serum, as
published previously (16): At neutral pH (7.0) and in the
presence of MgCl2, sulfated -cyclodextrin and dextran
sulfate form water-soluble complexes with LDL, VLDL, and chylomicrons
that are not accessible to PEG-modified enzymes. As HDL is not
complexed, its cholesterol moiety is readily available for enzymatic
quantification by using cholesterol esterase and cholesterol oxidase
coupled to PEG.
The aim of this study was to evaluate the new homogeneous HDL-C assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All analyses with the homogeneous and the precipitaton assays
were performed within 1 day of blood collection. Measurements of HDL-C
with the CDC reference method were performed in aliquots of the samples
kept at -20 °C for not longer than 4 weeks.
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Lipid measurements.
Cholesterol and triglycerides were
determined enzymatically with the CHOD-PAP and GPO-PAP method,
respectively. All reagents and control sera were obtained from
Boehringer Mannheim. All measurements were performed on Boehringer
Mannheim/Hitachi autoanalyzers types 704, 717, 747, 911, or 917.
Control sera Precinorm® L and Precipath® L
(both from Boehringer Mannheim) were included in each analytical run.
Homogenous HDL-C assay.
The reagents for the homogeneous
HDL-C assay were obtained from Boehringer Mannheim. The test was
performed according to the manufacturer's recommendations. In each
laboratory, the homogeneous HDL-C assay was calibrated with Precinorm
L, a lyophilized control serum in which HDL-C was determined by the
manufacturer with the PTA/MgCl2 precipitation procedure.
HDL-C determination by precipitation method.
All
laboratories used the PTA/MgCl2 method as the comparison
method. The reagents were purchased from Boehringer Mannheim. The test
was performed according to the manufacturer's recommendations.
HDL-C determination by combined ultracentrifugation and
precipitation method.
At laboratory 2 a combined
ultracentrifugation and precipitation assay was used as an additional
comparison method. Essentially, the protocol of the Lipid Research
Clinics Program was followed, with modifications previously described
(19)(20). In brief, VLDL was floated by
ultracentrifugation (d = 1.006 kg/L) and LDL was
separated from HDL in the infranatant by precipitation with
PTA/MgCl2.
HDL-C determination according to CDC reference method.
This comparison method, recommended by the National Cholesterol
Education Program (NCEP) Lipoprotein Measurement Working Group as the
accuracy base, was performed at laboratory 6 in addition to the
PTA/MgCl2 method (15). The laboratory is a
member of the Cholesterol Reference Method Laboratory Network
coordinated by the CDC. The CDC reference method includes removal of
VLDL by ultracentrifugation, precipitation of LDL from the bottom
fraction (d = 1.006 kg/L) with 46 mmol/L
heparin–manganese, and analysis of HDL-C in the resulting supernatant
by the CDC modified Abell–Kendall method
(19)(21).
Isolation of HDL
2 and
HDL3. HDL2 and HDL3
were isolated by sequential ultracentrifugation with 1.063<
d <1.125 kg/L and 1.125< d <1.21 kg/L as
density limits, respectively (22).
Linearity.
Testing of linearity was performed with the
Lin-Trol® linearity set for HDL-C from Sigma Diagnostics.
The HDL-C Lin-Trol was mixed with HDL-C Lin-Trol diluent at 11
different concentrations ranging from 0% to 100%. In addition, serum
from an apo A-I-deficient patient was supplemented with
HDL2 and HDL3 isolated by ultracentrifugation.
Lowest detectable concentration.
The lowest detectable
concentration was determined by 21 measurements by using isotonic NaCl
solutions as samples. From these results the mean + 3SD was calculated.
Bilirubin measurement.
Total bilirubin was measured
after its release from albumin with 2,5-dichlorophenyldiazonium salt as
the dye. The reagents were purchased from Boehringer Mannheim and
Merck.
Interferences.
Interference from hemoglobin was analyzed
according to Glick et al. (23). In addition, the
influences of hyperbilirubinemia and hypertriglyceridemia were analyzed
with the samples of all intermethod comparisons. The bias of HDL-C
(homogeneous HDL-C assay minus PTA/MgCl2 precipitation)
were plotted vs bilirubin.
Comparison of serum, lithium heparinate, and EDTA
plasma.
In five samples, the influence of lithium heparinate and
EDTA plasma in comparison with serum was investigated. Each sample was
measured five times.
Storage of samples.
HDL-C was determined in 50 fresh
serum samples by using the homogeneous assay. The samples were stored
at -20 and -70 °C for 4 weeks before the samples were reanalyzed.
Statistical methods.
Regression analyses were performed
with the method of Passing and Bablok (24). Precision data
were calculated according to recommendations of the NCCLS/EP5-T
protocol (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The within-day variance was determined as the average of the
variances obtained at each day. The between-day variance was calculated
from the variance of the means obtained each day, which was then
adjusted for the within-day variance component. The total precision CVs
ranged from 1.3% to 6.7%. Intraassay CVs ranged from 0.6% to 6.4%,
interassay CVs from 1.1% to 3.1%.
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In each run, two control samples were measured as duplicates. The results of the Precinorm L and Precipath L in laboratories 1 to 5 ranged from 94.3% to 105.5% (mean: 99%) and from 98.5% to 114.8% (mean: 103%) of the manufacturer's stated values (530 mg/L and 330 mg/L, respectively, as determined with the PTA/MgCl2 precipitation assay). The total CVs of the homogeneous HDL-C determined calculated from these samples were between 1.1% and 4.6%.
Linearity.
Results of the homogeneous HDL-C assay with
the Lin-Trol dilution experiments revealed linearity up to at least
1500 mg/L (y = 0.971x + 11.7 mg/L,
r = 0.9998). Similar results were obtained in
experiments in which HDL2 and HDL3, isolated by
sequential ultracentrifugation, were added at increasing amounts to
serum from a patient with apo A-I deficiency (HDL2:
y = 0.9911x + 39.6 mg/L, r =
0.9987; HDL3: y = 1.037x -
3.5 mg/L, r = 0.9996).
Lowest detectable concentration.
The lowest HDL-C
concentration that could be measured reliably with the homogenous assay
was ~30 mg/L.
Intermethod comparison.
The homogeneous HDL-C assay was
compared with a conventional PTA/MgCl2-based precipitation
method in all laboratories. A total of 1074 fresh unfrozen sera were
analyzed in parallel. Mean total cholesterol and total triglycerides
ranged from 1967 to 2639 and from 1702 to 2944 mg/L, respectively.
Maximum cholesterol and triglyceride concentrations were 8080 and
24 260 mg/L, respectively. HDL-C concentrations ranged between 12 and
1560 mg/L, determined with the PTA/MgCl2 method.
The correlation coefficients were between 0.956 and 0.994 (Table 3
). When we estimated the parameters of the regression lines
according to Passing and Bablok, slopes and intercepts ranged between
0.99 and 1.08 and -23.0 and 24.1 mg/L, respectively (24).
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In addition to the PTA/MgCl2 precipitation of native
sera, two different methods for HDL-C quantification involving
ultracentrifugation at d = 1.006 kg/L were
investigated. Table 3
and Fig. 1
A show that the combined ultracentrifugation and
PTA/MgCl2 precipitation method and the new homogeneous
HDL-C assay were strongly correlated. However, in some of the icteric
samples the homogeneous assay revealed HDL-C values lower than the
comparison method (see below).
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The new homogeneous HDL-C assay was in good agreement with the CDC
reference method as well (Table 3
, Fig. 1B
). None of the samples
analyzed with the CDC reference method was hypertriglyceridemic or
icteric.
Taken together, the results of the intermethod comparison suggest that the new homogeneous assay allows reliable determination of HDL-C.
Interferences.
Hemoglobin at concentrations up to 10 g/L
did not interfere with the new homogeneous HDL-C assay (data not
shown). We examined the results obtained in icteric samples of the
intermethod comparison study. This revealed that the homogeneous assay
produced low HDL-C in samples with bilirubin concentrations >100 mg/L,
compared with the PTA/MgCl2 precipitation (Fig. 2
).
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Specificity.
In Fig. 3
the bias of the homogeneous HDL-C assay minus HDL-C determined
by the combined ultracentrifugation and PTA/MgCl2
precipitation method are plotted against triglycerides, VLDL-C, and
LDL-C, respectively. Neither triglycerides, VLDL-C, nor LDL-C
systematically influenced the differences of the two HDL-C measurements
up to concentrations of 9000, 2600, and 3000 mg/L, respectively.
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Comparison of serum, lithium heparinate, and EDTA
plasma.
There were no significant differences between HDL-C
measured in either serum or heparin plasma: In four samples, deviations
were 
8.7 mg/L (data not shown). In one sample the deviation was 20.4
mg/L. The average bias between serum and heparinate plasma was -6.2
mg/L, the relative deviation being <1.9% in each case.
EDTA plasma yielded lower results compared with serum: The overall bias was -46.6 mg/L; the percentage deviations ranged from 4.7% to 11.5%.
Storage.
In 46 of 49 samples, excellent agreement was
obtained when the homogeneous assay was used to measure HDL-C in fresh
native sera and in sera stored at -20 and -70 °C for 2 and 4
weeks, respectively. The slopes ranged between 0.97 and 0.99 and the
intercepts were negligible. However, three samples showed deviations of
-60, 200, and 380 mg/L, respectively, compared with the results in
fresh native sera. The total cholesterol and the total triglyceride
content of these samples was <2500 mg/L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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So far, HDL-C has widely been determined by methods in which apo B-containing lipoproteins are precipitated with polyanions and bivalent cations. These methods are precise and agree well with ultracentrifugation methods (26)(27). However, they involve removal of the complexes of lipoproteins and polyanions by centrifugation, which is tedious and prevents full automation. Recently, some of us reported the evaluation of a homogeneous HDL-C assay that involves PEG and antibodies specific for apo B and apo C-III to exclude cholesterol of non-HDL particles from the enzymatic detection (17). The performance characteristics of that assay were satisfactory. However, because four different reagents were used, the assay was confined to the availability of special autoanalyzers. We now present the results of a multicenter evaluation of another homogeneous assay that overcomes this limitation because it involves two reagents only (16).
In all participating laboratories, the precision of this assay was better than the precision of the conventional precipitation methods (11)(12)(13). The NCEP performance goals for 1998 demand that the CV of HDL-C determinations be <4% at concentrations >420 mg/L (15), a criterion that was met by the new homogeneous HDL-C assay. The NCEP precision goal for HDL-C concentrations <420 mg/L is a SD <17 mg/L (15). In the human serum pools used for the determination of the imprecision the lowest HDL-C concentrations were 273 and 253 mg/L, respectively. The SDs for these HDL-C concentrations were 8.1 and 4.6 mg/L, respectively, and thus far below the permitted value of 17 mg/L. Thus, the NCEP precision goals will obviously be met by the homogeneous assay at both high and low HDL-C concentrations. Calculations of the total error were not performed because a preliminary calibration was used in this study. The definite calibration of the homogeneous assay, which will differ from that used here by <3%, will further diminish the differences between the homogeneous HDL-C assay and conventional methods to measure HDL-C (Cobbaert et al., submitted).
Comparison of the homogeneous HDL-C assay with a conventional PTA/MgCl2 procedure in 1074 samples produced ample correlation coefficients. The slopes of the regression lines ranged between 0.99 and 1.08 with small positive or negative intercepts, respectively. Therefore, in none of the participating centers did the mean HDL-C values of the homogeneous assay differ significantly from those of the PTA/MgCl2 procedure. The parameters of the regression lines were highly consistent throughout the different laboratories. This is in contrast to previous multicenter evaluations of methods for HDL-C determination and shows that the new homogeneous assay for HDL-C is robust (11)(12)(13)(14).
The homogeneous assay for HDL-C was compared with reference methods, including ultracentrifugation, in two laboratories. In laboratory 6 only normolipidemic samples were analyzed; this resulted in a correlation coefficient of 0.992 and a difference of the mean of 2 mg/L. Slope and intercept were not significantly different from 1 and 0, respectively. Similiar results were obtained between a combined ultracentrifugation and precipitation method in laboratory 2 if icteric samples were disregarded.
The accuracy of the homogenous HDL-C assay was not impaired by free hemoglobin up to 10 mg/L (23). In some samples bilirubin at concentrations of >100 mg/L brought about marked differences of HDL-C in the homogeneous assay, compared with the PTA/MgCl2 procedure. Supplementation experiments with ditaurobilirubin and unconjugated bilirubin revealed, however, that the PTA/MgCl2 method was more susceptible to these interferences than the new homogeneous assay (data not shown). The influence on HDL-C determined with both the homogeneous assay and the PTA/MgCl2 procedure was more pronounced with unconjugated bilirubin compared with ditaurobilirubin, respectively. Analyses of the data of laboratory 2 indicate that total triglycerides and VLDL-C did not affect the homogeneous HDL-C assay up to a triglyceride concentration of at least 8000 mg/L.
The homogeneous assay allows the use of lithium heparinated plasma
instead of serum. HDL-C measurements in EDTA plasma yielded lower
results than expected from the dilution introduced by the EDTA
solution, which reduces the results by ~3% compared with serum. This
may be due to the fact that the binding of -cyclodextrin to apo
B-containing lipoproteins depends on Mg2+ and that
EDTA, through its ion capturing capacity, reduces the availability of
Mg2+ (16). Another explanation could be the
osmotic effect of EDTA, which causes a shift of water from cells to
plasma, with a dilution of the plasma by ~3%.
Freezing of samples should be avoided. Our data show that in most samples freezing did not influence the results, but three outliers of 49 samples suggest that freezing may affect the results in particular situations. There was no obvious reason for the discrepancies in the three samples, as lipids and lipoproteins as well as the appearance of the samples themselves were absolutely normal. The addition of sucrose to serum might help to overcome this problem (28).
Because the assay is convenient to use in routine laboratories, costs have to be considered. The reagents for the homogeneous HDL-C assay are approximately fivefold more expensive than conventional precipitation reagents for HDL-C. However, total assay costs depend on many other aspects, such as the number of HDL-C determinations per day, materials needed for the pretreatment of samples (conventional assay), type of dilutor (manual or automated), and the time of the technical assistant to perform the analysis. In addition, the possibility of errors such as misidentification or loss of the sample (during centrifugation) is much higher with the conventional HDL-C assay. A further advantage of the new homogeneous HDL-C assay is the small sample volume needed. We are convinced that in most laboratories the higher reagent costs will be compensated by economizing the laboratory work and that the homogeneous HDL-C assay will improve the accuracy of the HDL-C determination.
In summary, the new homogeneous assay produces precise and accurate determinations of HDL-C. Even hypertriglyceridemic samples up to at least 8000 mg/L and samples with bilirubin <100 mg/L showed unbiased results. Thus this homogeneous assay represents a significant improvement of our methodology to quantify HDL-C and may facilitate the identification of individuals at increased risk of atherosclerosis.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cyclodextrin. Clin Chem 1995;41:717-723.
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, normal samples; 
,
icteric samples (bilirubin >50 mg/L).
