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Articles |
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School of Allied Health Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
2
Department of Epidemiology and Mass Examination for CVD,
Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3
Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
3
Department of Clinical Chemistry, SRL Inc., 51
Komiya-cho, Hachiohji-shi, Tokyo 192-8535, Japan.
4
Laboratory of Chemistry, College of Liberal Arts and
Sciences, Tokyo Medical and Dental University, 2-8-30, Kohnodai,
Ichikawa-shi, Chiba 272-0827, Japan.
a Author for correspondence. Fax 81-47-300-7100; e-mail okazaki.cul{at}cul.tmd.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used four HPLC instruments with different column-load numbers from 250 to 5000. For accuracy assessment of TC and HDL-C, we used the reference methods recommended by the CDC.
Results: The values measured by the four instruments were highly correlated with each other (mean r = 0.965), and the absolute mean differences were 4–43 mg/L for TC, 4–30 mg/L for HDL-C, 0–48 mg/L for LDL-C, 7–66 mg/L for VLDL-C, 0–7 mg/L for CM-C, 0.1–0.3 nm for LDL size, and 0–0.1 nm for HDL size. For TC, the HPLC instruments showed high correlation and good agreement with the reference method: r = 0.997; total error <6.6%; absolute mean bias <1.2%. For HDL-C, the results from the HPLC method were significantly higher (10.8% absolute mean bias) than those of the CDC reference method, in spite of good correlation between the two methods (r = 0.998).
Conclusions: The between-instrument variation in serum lipoprotein analysis by HPLC was confirmed to be very small. This method met the US National Cholesterol Education Program’s performance criteria for TC but not for HDL-C.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lipoprotein particles are classified on the basis of their hydrated density, size, and electrophoretic mobility. Major lipoprotein classes have been defined according to their density. The separation and quantification of lipoproteins by ultracentrifugation is not suited to the clinical laboratory setting, however, because the method is time-consuming and requires special training and large sample volumes.
HPLC is a method for classifying and quantifying lipoproteins by particle size (6). In the HPLC method, the lipoproteins separated by gel permeation columns (TSKgel LipopropakXL; Tosoh) are detected by an on-line enzymatic reaction for cholesterol. Chylomicron cholesterol (CM-C), VLDL-cholesterol (VLDL-C), LDL-C, HDL-C, and total cholesterol (TC) are calculated automatically by a computer (7). The greatest advantages of the HPLC method are direct profiling of lipoproteins in small sample volumes, and rapid and simultaneous determination of their size distribution. For many epidemiological and clinical studies, a reliable and convenient technique is required to allow quantification and characterization of lipoprotein subclasses directly from serum samples.
Several studies have compared the analytical performance of the HPLC method for lipoproteins with established methods, including an automated enzymatic assay for TC (8), and homogenous (9)(10), precipitation (9)(10)(11), and sequential ultracentrifugation (7) methods for HDL-C. However, the interlaboratory or between-instrument variation of this assay has not been examined. Furthermore, it is necessary to assess the accuracy of the HPLC method in comparison with the reference method to establish traceability to the reference method recommended by CDC (12).
The main purpose of this study was to evaluate the between-instrument variation for measurement of TC, HDL-C, LDL-C, VLDL-C, CM-C, LDL size, and HDL size. We also participated in the certification protocols provided by the CDC’s Cholesterol Reference Method Laboratory Network to assess the accuracy of the TC and HDL-C assays.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the TG interference study, serum samples (group C) were collected from hypertriglyceridemic patients (n = 19) with serum TG concentrations >2000 mg/L at Osaka University Hospital after informed consent. Group C samples were kept at 4 °C and analyzed within 10 days after sample collection.
Precision studies were performed using two different pooled sera (QC1 and QC2) stored at -80 °C. QC1 and QC2 were prepared by the same procedure as described above and had TC values of 1370 and 2180 mg/L, respectively.
calibration material
An in-house pooled serum (lot no. 90920) was prepared as the
calibration material for the HPLC method. This material was obtained by
combining fresh sera from healthy volunteers after a 12-h fast.
Reference values (1770 mg/L for TC and 653 mg/L for HDL-C) were
assigned to this material, using the reference methods at OMC. Aliquots
of the material were stored at -80 °C.
hplc method
We used an automated TOSOH Lipoprotein Analytical System (Tosoh),
which included an AS-8020 autosampler, CCPS and CCPM-2 pumps,
and a UV-8020 detector (7). Serum lipoproteins were
separated using two connected columns (300 x 7.5 mm) of TSKgel
LipopropakXL (Tosoh) with TSKeluent LP-1 (Tosoh) as the elution buffer
at a flow rate of 0.700 mL/min. Cholesterol was detected by an on-line
enzymatic reaction through the mixing of the effluent from the columns
with a commercial kit, Determiner L TC (Kyowa Medex). Reagents 1 and 2
of the commercial kit were continuously pumped at flow rates of 0.263
and 0.087 mL/min, respectively. The absorbance at 550 nm was
continuously monitored after the enzymatic reaction at 45 °C in a
reactor coil (7.5 m x 0.4 mm i.d.). A 5-µL sample was injected
at an interval of 16 min per sample. An in-house pooled serum (lot no.
90920) was used as the calibrator. HPLC profiles of the calibration
material and a sample from a hyperlipidemic patient (TC, 3240 mg/L; TG,
14 460 mg/L) are shown in Fig. 1
. The cholesterol concentrations in the major lipoprotein
classes were estimated from a linear calibration curve of the
assigned TC value plotted against the total area with the
calibrator, after automatic calculations of total area and individual
areas corresponding to HDL, LDL, VLDL, and CM.
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Calibration of columns was carried out using latex beads (Magsphere) 25 and 37 nm in diameter, and high-molecular weight calibrators (Pharmacia Biotech) containing thyroglobulin (17 nm), ferritin (12.2 nm), catalase (9.2 nm), albumin (7.1 nm), and ovalbumin (6.1 nm). The mean particle sizes of LDL and HDL in the calibrators were 25.3 and 11.3 nm, respectively, based on the calibration curve. The elution times of the observed peaks on a chromatogram were converted to particle sizes, using a linear calibration curve of the logarithm of the particle sizes plotted against the elution times corresponding to LDL and HDL in the calibrator.
In this study, we used four HPLC systems (instruments A, B, S1, and S2) with different column-load numbers. The term "column-load number" indicates the number of samples injected into the columns. At the beginning of this study, the column-load numbers for each instrument were ~5000 (column nos. T9056 and T9057) for instrument A, 250 (column nos. 84SM2Y0005 and 84SM2Y0006) for B, 2000 (column nos. 84SM1Y0023 and 84SM1Y0024) for S1, and 2500 (column nos. T9091 and T9092) for S2. The HPLC method was performed at Tokyo Medical and Dental University with instruments A and B (in Tokyo and Chiba, respectively), and at SRL Inc., in Tokyo, with instruments S1 and S2. The same calibrator was used for all instruments. All samples were analyzed in duplicate.
cdc reference methods for tc and hdl-c
The reference method for cholesterol is the
Abell-Levy-Brodie-Kendall assay, as modified by the CDC, described
previously in detail (13). In this method, serum is
saponified with ethanolic KOH and extracted with hexane. An aliquot of
the extract is evaporated, and the residue is reacted with
Liebermann-Burchard reagent to develop the color, which is measured at
620 nm.
The reference method for HDL-C consists of a three-step procedure involving ultracentrifugation, precipitation, and cholesterol analysis (14). After triglyceride-rich lipoproteins are removed from the serum by ultracentrifugation against a solution with a density of 1.006 kg/L, apolipoprotein B-containing lipoproteins in the ultracentrifugal infranate are precipitated with heparin and MnCl2, and the cholesterol in the heparin-MnCl2 supernatant is measured by the Abell-Kendall method at CDC.
The reference methods were performed in duplicate at OMC in Japan.
tg analysis
TG values were determined enzymatically in a single analysis with
a Hitachi 7170 automated analyzer, using reagent kit L Type Wako TG-H
(Wako Pure Chemical Industries). The TG assay was corrected for the
presence of endogenous glycerol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Two different materials (QC1 and QC2) with low (1370 mg/L) and
high (2180 mg/L) TC concentrations were analyzed to assess the
precision of the four HPLC systems. One aliquot of each pooled serum
was assayed by each instrument in five replicates each day for 4 days
and in duplicate each day for 16 days. The within-day variance
(Vwithin) was determined as the mean of
the daily variances at the first 4 days. The between-day variance
(Vbetween) was determined as the variance (V) of
the daily means at 20 days, which was adjusted for the within-day
variance component; Vbetween = V -
(Vwithin/Number of repeats per day).
Total variance (Vtotal) was the value of
Vwithin + Vbetween.
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At the low TC concentration (QC1), the total imprecision (CV) values for A, B, S1, and S2, respectively, were as follows: 1.5%, 2.7%, 1.6%, and 2.0% for TC; 1.9%, 3.3%, 2.0%, and 2.4% for HDL-C; 1.2%, 2.2%, 1.6%, and 1.4% for LDL-C; 2.3%, 5.2%, 4.5%, and 5.4% for VLDL-C; 12%, 19%, 43%, and 32% for CM-C; 0.35%, 0.37%, 0.39%, and 0.33% for LDL size; and 0.55%, 0.92%, 0.82%, and 0.43% for HDL size. At the high TC concentration (QC2), the total imprecision values for A, B, S1, and S2, respectively, were as follows: 1.7%, 1.8%, 0.8%, and 0.8% for TC; 2.8%, 2.8%, 0.9%, and 1.2% for HDL-C; 1.8%, 1.9%, 1.3%, and 1.5% for LDL-C; 2.5%, 4.7%, 4.2%, and 5.9% for VLDL-C; 17%, 11%, 37%, and 29% for CM-C; 0.37%, 0.39%, 0.50%, and 0.31% for LDL size; and 0.73%, 0.78%, 0.79%, and 0.41% for HDL size. Because the CM-C concentrations in the samples used were <7 mg/L, the CVs were relatively higher than for the other analytes; the SD for the CM-C assay was 0.4–1.7 mg/L.
between-instrument comparison
Using four HPLC instruments and group-A samples (n = 50), we
estimated between-instrument variation for the measurement of
lipoprotein cholesterol concentrations (TC, HDL-C, LDL-C, VLDL-C, and
CM-C) and the particle sizes of LDL and HDL. To examine the degree of
agreement among the values obtained by the four instruments, we
calculated linear regression equations by Deming regression analysis
and the absolute mean differences between each apparatus (Table 2
). All four instruments correlated highly with each other and
produced good correlation coefficient values of 0.996–0.999 for TC,
0.978–0.995 for HDL-C, 0.992–0.998 for LDL-C, 0.977–0.995 for
VLDL-C, 0.962–0.984 for CM-C, 0.884–0.947 for LDL size, and
0.887–0.931 for HDL size. The absolute mean differences among HPLC
apparatuses were 4–43 mg/L for TC, 4–30 mg/L for HDL-C, 0–48 mg/L
for LDL-C, 7–66 mg/L for VLDL-C, 0–7 mg/L for CM-C, 0.1–0.3 nm for
LDL size, and 0–0.1 nm for HDL size.
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Statistical differences as assessed by the Student paired t-test were not observed between the TC values obtained from instruments S1 and S2, between the HDL-C values obtained from B and S2, between the LDL-C values obtained from B, S1, and S2, and between CM-C values obtained from A and S2. For the VLDL-C, LDL size, and HDL size assays, the differences between the values obtained from the four instruments were significant (P <0.05) but slight. LDL and HDL were detected in all samples by all four instruments, but CM was detected in only 15 samples by A, 24 samples by B, 12 samples by S1, and 16 samples by S2. VLDL was not detected by any of the instruments.
Between-instrument imprecision values, which were calculated by an analysis of variance, were 1.9% for TC, 3.1% for HDL-C, 3.0% for LDL-C, 10% for VLDL-C, 49% for CM-C, 0.6% for LDL size, and 0.8% for HDL size.
accuracy for tc and hdl-c
The results obtained for the TC assay with all four instruments
and the reference method are shown in Fig. 2
and Table 3
. Linear regression analysis by the least-squares method showed
that only instrument B gave a slope that was significantly >1.0, but
the intercepts did not differ significantly from 0 mg/L for any of the
instruments. The estimated values at 2200 mg/L (the upper end of the
reference interval), using the regression lines, were 2212 mg/L
for A, 2222 mg/L for B, 2189 mg/L for S1, and 2195 mg/L for S2, which
corresponded to 0.5%, 1.0%, -0.5%, and -0.2% bias, respectively.
The mean biases for A, B, S1, and S2 were 0.3%, 1.2%, -0.6%, and
-0.5%, respectively. The total error (%), which is equal to the
systematic error [absolute mean bias (%)] plus the random error
[total imprecision (%) x 1.96], was 3.6% for A, 6.6% for B,
3.7% for S1, and 4.3% for S2 (Table 3
).
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Three instruments (A, B, and S1) were used for the comparison with the
reference method for HDL-C. Group B samples (n = 46) were analyzed
using the same conditions as for the TC assay. The results are shown in
Fig. 3
and Table 4
. Although the two methods correlated very well, with
correlation coefficients of 0.996–0.999, the slopes and intercepts of
the regression lines were significantly different from 1.0 and 0
mg/L, respectively. The HPLC method overestimated HDL-C values at all
points between 208 to 853 mg/L when compared with the reference
method. Estimated values at 350 mg/L (the lower end of the
reference interval), using the regression lines, were 396 mg/L (13.1%
bias) for A, 381 mg/L (8.9% bias) for B, and 395 mg/L (12.9% bias)
for S1. Mean biases of 12.3%, 8.4%, and 11.6% were found for
instruments A, B, and S1, respectively, for the HDL-C assay. The total
error was 17.2% for A, 15.0% for B, and 13.6% for S1.
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tg interference
We used instrument A and group C samples (n = 19) to assess
the performance of the HPLC method for patients with a wide range of
serum TG concentrations from 2110 to 53 200 mg/L. With one sample that
had a TG concentration of 53 200 mg/L, the TC value was remarkably
overestimated by the HPLC method relative to the reference method: the
TC values determined by the reference and HPLC methods were 2945 and
4306 mg/L, respectively. Because the bias of 46.2% (1361 mg/L)
exceeded the sum of the mean bias plus SD multiplied by 3, the sample
was excluded from all data analyses, although the reason for the
discrepancy was not clear.
In Fig. 4
A, the reference (x) and HPLC (y) methods
yield a linear regression line of y = 1.06x
- 152 (r = 0.951; n = 18) for TC assay. Fig. 5
A, in which the biases vs the reference method are plotted as a
function of serum TG concentrations, shows an increasingly negative
bias (r = -0.743; P <0.001) with
increasing TG concentration. Samples from patients with high TG
concentrations often give high VLDL-C and/or high CM-C values. Plotted
as a function of CM-C concentration as determined by the HPLC method
rather than serum TG concentrations, TC assay biases were inversely
correlated (r = -0.963: P <0.001) with
CM-C concentrations (Fig. 5B
). Therefore, we assumed that the detection
of CM-C might be incomplete in the HPLC method, which would lead to
underestimation of TC. The mean bias, however, was 0.1% (6 mg/L) and
small in an overall range of TG concentrations up to ~17 000 mg/L.
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For the HDL-C assay, the reference (x) and HPLC
(y) methods were highly correlated (r =
0.985) and produced a linear regression line of y =
1.04x + 52.9 (n = 18), as shown in Fig. 4B
. The HPLC
method gave a mean bias of 20.0% vs the reference method for group C
samples, and the bias was positively higher than for samples with TG
concentrations within the reference interval. Fig. 5C
shows the
HDL-C assay biases plotted as a function of serum TG concentrations,
suggesting that there is correlation (r = 0.720;
P <0.001) between HDL-C assay bias and TG concentration. We
also compared the HDL-C values obtained by the HPLC method from samples
before and after ultracentrifugation at OMC (Table 5
). Although statistical analysis by the Student paired
t-test gave a significantly higher (not lower) result after
ultracentrifugation than before ultracentrifugation, the mean
difference (HDL-C results after ultracentrifugation minus HDL-C results
before ultracentrifugation) was very small (16 mg/L). If TG-rich
lipoproteins of d <1.006 kg/L had made the HPLC method
overestimate the HDL-C values, the results after ultracentrifugation
would have been lower than those before ultracentrifugation. The HPLC
method for HDL-C, therefore, was considered less affected by TG-rich
lipoproteins.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the precision study, the four HPLC systems met the performance goals for measurement of TC (CV <3%), HDL-C (CV <4% at HDL-C >420 mg/L), and LDL-C (CV <4%) required by the recent National Cholesterol Education Program (NCEP) guidelines (18)(19). It was, however, impossible to assess the precision of measurements of VLDL-C, CM-C, and particle sizes by the HPLC method because at present there are no NCEP guidelines for these assays. Our results did reveal that the HPLC method produced better precision (mean CV = 4.6%) for the VLDL-C assay than the single vertical spin auto profiler (CV = 4.8%) (20) and fast lipoprotein chromatography (CV = 5.8%) (21).
In our between-instrument comparison study, a small variation was found in determinations of lipoprotein cholesterol concentrations and particle sizes. However, we did not find a tendency for column-load numbers to have a direct effect on the lipoprotein profile. The precision remained high for column-load numbers in a wide range from ~250 to 5000.
According to the NCEP guidelines for TC measurements, the goals for
total error and absolute mean bias are 
8.9% and 3%, respectively
(19). The results in the accuracy study revealed that all
HPLC systems, with columns with different column-load numbers, met the
NCEP performance criteria for the TC assay when the same calibrator
(lot no. 90920) was used, and suggested that the recovery of
lipoproteins from columns was satisfactory. The NCEP
performance goals for HDL-C require a total error of 13% and an
absolute mean bias of 5% (18). These criteria were not
met by three HPLC systems, which had total errors 
13.6% and absolute
mean biases 8.4%. Considering the high precision of the HPLC method
for HDL-C (CV 3.3% at HDL-C >420 mg/L), the disagreement between
the reference and HPLC methods must have been caused by systematic
error, not random error.
The ratio of HDL-C to TC in the calibrator was 36.9%, calculated from the reference values, whereas the ratio of HDL area to total area of the HPLC pattern obtained by the four instruments was 38.6% on average. All HPLC systems overestimated HDL-C in the calibrator, and this positive bias can in part explain the systematic error. When we calculated the HDL-C concentration using a linear calibration curve of the assigned HDL-C value plotted against the area corresponding to the HDL in the calibrator, the absolute mean bias was <5.2% instead of 12.3%.
Although we confirmed that the traceability of the HPLC method to the reference method for HDL-C was improved by use of the reference material mentioned above, the problem remains as to whether the HDL classified by HPLC is the same as the HDL classified by the reference method. The HDL separated by the HPLC may be different from that in the supernatant obtained after precipitation with the reference method. HDL particles are heterogeneous in density, size, charge, and apolipoprotein component. We have reported previously that the phosphotungstic acid/MgCl2 precipitation assay underestimated HDL-C values relative to the HPLC method because of the high concentration of MgCl2 in a commercial kit, whereas a homogeneous assay produced values close to the HPLC method (10). On the other hand, heparin-Mn2+ reagent is not likely to precipitate HDL subclasses such as apolipoprotein E-containing HDL particles (22). Further studies with individual samples will be required to answer this question.
When TG concentrations were increased, the TC assay bias was
acceptable, at 3%, with some exceptions: the HPLC method became
negatively biased, especially in the presence of increasing CM-C
concentrations. This finding indicates that the determination of CM-C
by the HPLC method is incomplete, probably because of inadequate
enzymatic detection of CM-C. On the other hand, HDL-C concentrations
were significantly overestimated by the HPLC method compared with the
reference method, and the positive bias was correlated with serum TG
concentration. Ultracentrifugation to remove TG-rich lipoproteins prior
to analysis, however, did not eliminate this problem. We therefore
concluded that the HPLC method for HDL-C was affected only minimally by
the presence of TG-rich lipoproteins.
Methods based on different principles will produce different values. Wiebe and Smith (23) described how it might be possible to adjust reagent concentrations, ionic strength, buffer pH, or other characteristics of the HDL isolation reagents so that the HDL-C values would mimic those obtained with another method. It is important in clinical sites that values obtained by new methods be close to the target values. In terms of standardization of HDL-C measurement, the accuracy should be based on the CDC reference method, which has yielded coronary heart disease risk estimations and population distributions.
In summary, the between-instrument variation in serum lipoprotein
analysis by the HPLC method was confirmed to be very small. This method
met the NCEP criteria for the TC assay. As for the HDL-C assay, the
values obtained with the HPLC method were significantly higher than
those obtained with the reference method, in spite of good correlation
between the two methods. The reason for this difference is not clear at
present. However, the HPLC method gives considerable information, such
as size distribution of heterogeneous lipoproteins, in a chromatogram,
as shown in Fig. 1
. We believe that additional information that other
methods do not provide is present on the chromatogram. Analysis
of the chromatogram for both quantitative and qualitative information
may be useful for studies on the serum lipoproteins that play a vital
role in the development of atherosclerosis.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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G. R. Warnick, M. Nauck, and N. Rifai Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays Clin. Chem., September 1, 2001; 47(9): 1579 - 1596. [Abstract] [Full Text] [PDF]
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 Y. Ishigaki, S. Oikawa, T. Suzuki, S. Usui, K. Magoori, D.-H. Kim, H. Suzuki, J. Sasaki, H. Sasano, M. Okazaki, et al. Virus-mediated Transduction of Apolipoprotein E (ApoE)-Sendai Develops Lipoprotein Glomerulopathy in ApoE-deficient Mice J. Biol. Chem., September 29, 2000; 275(40): 31269 - 31273. [Abstract] [Full Text] [PDF]
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), S1 (
), and S2 (+).
The dotted line in A represents
y = x; the dotted
line in B represents y = 0.
Bias (%), [(HPLC - Reference method)/Reference
method] x 100.
