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Lipids and Lipoproteins |
a Author for correspondence. Fax 34-77-31-25-59; e-mail lab-hreus{at}grupsgs.com.
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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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1.6 mmol/L is considered protective
(3). The National Cholesterol Education Program (NCEP)
recommends that HDL-C be measured in all adults at their initial
screening (3)(4). Hence, there is an increasing
demand in clinical laboratories for reliable and easy-to-perform
methods for determining HDL-C. The reference method for HDL-C measurement is preparative ultracentrifugation (5), which is a cumbersome, time-consuming, and expensive technique. In most clinical laboratories, HDL-C is routinely measured by precipitation methods that are simpler and are in excellent agreement with those of ultracentrifugation (6)(7). However, these methods involve manipulation of the sample (a centrifugation step and recovery of supernatant), which precludes the full automation that is often a prerequisite for a high throughput clinical chemistry laboratory.
Recently, several reports (8)(9)(10)(11)(12)(13)(14)(15) have described homogeneous (direct) assays for HDL-C that are readily adaptable to automated analyzers, such that laboratories can have HDL-C determination as an on-line procedure for the routine screening of large populations. However, the application of these assays to specific, well-defined groups of patients has not been sufficiently investigated to date (16), a lack that has considerable importance in terms of efficient patient management.
The present study was aimed to: (a) evaluate a new homogeneous HDL-C assay; and (b) compare this assay with a well-accepted precipitation method in a range of clinically healthy subjects and in the elderly, as well as in patients with cardiovascular disease, nephrotic syndrome, or liver cirrhosis, pathologies in which lipoprotein profiles/concentrations are profoundly altered.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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procedures
Direct HDL-C assay.
The direct assay of HDL-C was performed
with commercial reagents obtained from Daiichi (Tokyo, Japan) and
supplied in Spain by ITC Diagnostics (Izasa, Barcelona, Spain). The
assay requires two reagents. In the first step, 2 µL of plasma are
incubated for 3.5 min with 210 µL of a mixture of polymers and
polyanions that block, by adsorption, the apoprotein B-containing
lipoprotein particles. In the second step, the noncomplexed cholesterol
is measured by the cholesterol oxidase-peroxidase (CHOD-PAP) method.
HDL-C determination by precipitation.
HDL-C was measured by
precipitation with polyethylene glycol (17). The
precipitating reagent was 95 g/L polyethylene glycol (molecular weight,
20 000) dissolved in phosphate buffer (0.033 mol/L
NaH2PO4 and 0.066 mol/L
Na2HPO4, pH 6.5). In this technique, 500 µL
of precipitating reagent is added at 4 °C to 500 µL of plasma. The
mixture is vigorously mixed for 15 s and centrifuged at
3000g at 4 °C for 20 min to pellet the apoprotein
B-containing lipoproteins. HDL-C was measured in the supernatant by the
CHOD-PAP technique. The intra- and interassay CV values were <3% and
<5%, respectively. In a preliminary assessment, we observed that the
values obtained by this technique did not differ significantly from
those obtained by sequential preparative ultracentrifugation in control
subjects (r = 0.90; y =
1.1x - 0.1; n = 30) nor in cirrhotic patients
(r = 0.89; y = 0.9x; n
= 37).
Other methods.
Cholesterol and triglycerides were determined
enzymatically with the CHOD-PAP and the lipase/GPO/PAP methods,
respectively; albumin was determined by the bromcresol green method;
bilirubin was determined by the Jendrassik and Gróf technique
(ITC Diagnostics, Barcelona, Spain); IgG and IgM were determined by
immunoturbidimetry (Biokit, Barcelona, Spain); hemoglobin was
determined in a Coulter® MD-10 counter (Coulter).
standard and control materials
Initially, two commercial HDL-C calibrators, one from ITC
Diagnostics and the other from Sigma Diagnostics® (EZ
HDL(TM) calibrator, Sigma Chemicals) were evaluated against purified HDL
obtained by ultracentrifugation (18). The calibrator from
ITC Dignostics gave cholesterol values ~20% higher than those
reported by the manufacturers, thus undervaluating HDL-C results when
used as a standard. Conversely, the cholesterol value of the Sigma
calibrator was identical to that reported in the commercial leaflets;
for this reason, the Sigma calibrator was the standard used in the
present evaluation. Lipid-Trol-TL Control Levels 1, 2, and 3
(Dade®, Düdingen, Switzerland) and three pools of
sera (concentrations established by direct method) designated as
"low" (0.61 ± 0.01 mmol/L), "normal" (1.13 ± 0.01
mmol/L), and "high" (1.99 ± 0.02 mmol/L) were used as quality
controls.
samples
Blood samples were selected from 98 control subjects (group A;
ages, 24–55 years) and from 240 assorted other individuals: 85 elderly
who were clinically healthy (group B; ages, 72–93 years); 93 patients
who had had at least one episode of acute myocardial infarction (AMI)
before the age of 55 years (group C; actual ages, 57–65 years); 25
patients presenting with a nephrotic syndrome (group D; ages, 26–64
years); and 37 patients with liver cirrhosis (group E; ages, 45–71
years). To preclude hormonal status effects on the measurements, only
blood samples from men were used in the present study.
The control subjects were randomly chosen from the routine health and safety-at-work checks conducted in several industrial companies in our area. Excluded were those with clinical or laboratory evidence of diabetes, neoplasia, renal disease, hepatic damage, and cardiovascular disease. Group B subject samples were from routine health monitoring in several residences for the elderly in the area. Patient samples for groups C, D, and E were from the outpatient clinics of the Hospital Universitari de Sant Joan de Reus. All procedures were in accordance with the ethical standards of the Ethics Committee of Hospital Universitari de Sant Joan, and anonymity of results was guaranteed. All blood samples were drawn in the fasted state into EDTA-containing glass tubes; the plasma was separated by centrifugation at 1500g for 15 min and stored at -20 °C for batched analysis.
performance evaluation of the direct hdl-c measurement
The direct HDL-C assay was evaluated as described elsewhere
(19).
statistical analysis
Mean values for HDL-C by the two methods were compared by
Student's t-test. Statistically significant differences
were set at P <0.05. Results are presented as means
± SD. The association between variables was measured by linear regression
analysis. The degree of agreement between the direct and the
precipitation methods was estimated by the Bland–Altman graphical
procedure (20) as described by Hollis (21). In
this method, a scattergram is drawn with the means of the results
obtained by the two procedures plotted against the differences between
them. Bias is noted as a lack of symmetry of these differences around
the value zero, and the limits of agreement are given by the mean
difference ± 2 SD of the differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total error.
Total error was calculated by adding the
systematic error and the random error, as previously described
(22). Results were as follows: 11.6% for Lipid-Trol-TL
Control Level 1; 11.3% for Level 2, and 6.5% for Level 3.
Accuracy.
Accuracy was estimated by adding 100 µL of normal
pool serum to identical volumes of the three commercial controls. The
percentage recoveries (calculated as measured HDL-C/theoretical
HDL-C x 100) from sextuplicate measurements were 98.7%
± 2.0% for Level 1, 104.0% ± 4.6% for Level 2, and
101.7% ± 1.6% for Level 3.
Linearity and detection limit.
Linearity was assessed by
quintuplicate measurements on serial dilutions of the high pool (from
undiluted to up a 1:32 dilution with physiologic saline). The
regression line of observed vs expected values was y =
0.99x 0.04 (r = 0.9995). To determine the
detection limit, the absorbance of the reagent blank was measured 20
times, the mean ± SD calculated, and the detection limit defined
as the HDL-C concentration corresponding to an absorbance equal to the
mean of the reagent blank value 2 SD. The detection limit thus
calculated was 0.10 mmol/L.
Interferences.
Assessment of interference from triglycerides,
hemoglobin, and bilirubin was performed as previously described
(23). The low, normal, and high sera pools were supplemented
with chylomicrons, hemoglobin, or bilirubin at various concentrations.
Results are shown in Fig. 1
. There was no substantial interference from triglycerides up to
40 mmol/L nor from bilirubin up to 150 µmol/L, but higher bilirubin
concentrations produced a marked decrease in the direct HDL-C
measurement. Hemoglobin interference strongly decreased the HDL-C value
and would suggest that the assay is invalid for hemolyzed samples.
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Effect of paraproteinemia.
The effect of nonspecific
paraproteinemia in HDL-C determination was assessed as the percentage
recovery after the addition to the low pool of three serial dilutions
of sera from two patients with IgG (47.9 g/L) and IgM (17.6 g/L)
myeloma. Results are shown in Table 2
. The recovery was not influenced by the paraprotein
concentration.
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Stability study.
Aliquots of the low, normal, and high pools
stored at -20 °C were periodically thawed over 4 months. The HDL-C
values did not vary significantly (P >0.05) over this
period (Fig. 2
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comparison between direct and precipitation methods
Values for HDL-C obtained by the precipitation and by the
homogeneous assays are shown in Table 3
. Results were practically identical in the samples from control
subjects and from the elderly. The homogeneous assay gave results
somewhat lower in samples from patients with AMI (difference between
means, -2.8%). However, these differences did not reach statistical
significance. Differences between means were higher and statistically
significant in samples from patients with the nephrotic syndrome
(-7.4%; P <0.05) and were particularly high in the
samples from patients with liver cirrhosis (-20.8%, P
<0.001). Pearson correlation coefficients comparing the two methods
were highly significant (P <0.001; Figs. 3
and
4, top panels). Tighter regressions were observed for control and
elderly groups (r = 0.90 and 0.95, respectively) than
for those of the AMI, nephrotic, and cirrhotic patient groups
(r = 0.76, 0.69, and 0.70, respectively).
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However, because correlation coefficients are measures of the
association between two methods but not of the agreement between them
(21), the degree of agreement was assessed using the
Bland–Altman (20) graphical technique (Figs. 3
and 4
,
bottom panels). These graphs are the differences between the two
methods (y-axis) plotted against the average between them
(x-axis). Agreement is indicated by the calculated value of
the bias derived from the mean and SD of the differences.
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For samples from control subjects, the mean difference was -0.01 mmol/L, which indicated that the homogeneous assay was in concordance with the precipitation method and did not systematically under- or overestimate HDL-C values. From this group, four samples (4.1%) had values >2 SD above or below the mean, i.e., outside the limits of agreement (21).
The same plot for samples from the elderly group is shown in Fig. 3
(bottom panel, right). As with the samples from the control subjects,
the mean difference was also close to zero (-0.01 mmol/L), but the
differences would seem to be dependent on the HDL-C concentration
because there appeared to be an overestimation by the homogeneous
technique at low HDL-C concentrations and an underestimation at values
>1.5 mmol/L. Two samples (2.3%) were outliers with discrepancies
>2 SD above or below the mean.
The degree of agreement between both techniques was worse in the other
groups of patients (Fig. 4
, bottom panels). In the samples from
subjects who had experienced an AMI (left panel), the mean difference
was close to zero (-0.03 mmol/L), but the limits of agreement were
very high, i.e., values showed a considerable degree of dispersion
above or below the mean. In the nephrotic syndrome samples (middle
panel), the degree of disagreement was similar (mean difference, -0.09
mmol/L). The worst results were observed in samples from patients with
liver cirrhosis, in whom a clear underestimation was noted when the
homogeneous assay was used. The mean difference was very low (-0.26
mmol/L), as were the limits of agreement (from -1.04 to 0.50 mmol/L).
Interestingly, the disagreement between the homogeneous and the
precipitation techniques in samples from patients with liver cirrhosis
seems to be associated with the degree of liver damage. As seen in Fig. 5
, bias plots show that the differences between the methods
depend on serum albumin and bilirubin concentrations, used in this
evaluation as markers of liver-function impairment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the three HDL-C concentrations studied, the assay fulfilled the NCEP total-error goal for 1998 of <13% (25). Our results differed those of Huang et al. (13), who, also using a Daiichi reagent, found very high total errors. A possible explanation for this discrepancy is the high systematic error they obtained when using the original calibrator material supplied by the manufacturers. Indeed, our results showed that the cholesterol concentration reported for this calibrator was too low and gave values that were 20% higher when assayed against purified HDL. This systematic error was resolved when a different standard material was used. These results would suggest that the accurate preparation of HDL-C standards could be a general problem that manufacturers of these particular materials ought to seriously address.
Accuracy and linearity were excellent. The detection limit was 0.10 mmol/L, which is sufficient to measure HDL-C at low concentrations reliably.
Lipemia up to a triglyceride concentration of 40 mmol/L appeared to have little influence on the assay. Bilirubin up to 150 µmol/L had no influence on the accuracy, but higher values induced a negative interference. This effect has been reported for other HDL-C assays (11)(13)(22) and would seem to be a function of cholesterol measurements in general (26). Paraproteins did not cause any interference with the assay. Hemoglobin markedly decreased the values obtained for HDL-C, which is surprising because this effect has not been reported for other, equivalent homogeneous assays (11)(13). Although hemoglobin has been shown to interfere with enzymatic cholesterol determinations (26), the presence of free hemoglobin could lead in the present assay system to alterations in matrix formation, which is the basis of the direct assay. Again, we differ from Huang et al. (13), who described a different interference pattern of the Daiichi reagent (negative interferences by bilirubin and triglyceride but not by hemoglobin). We cannot offer any explanation for this discrepancy except to suggest, perhaps, a qualitative modification in the manufacture of the reagent subsequent to that article.
Storage of samples at -20 °C for up to 4 months had no effect on the measurements of HDL-C and is an advantage for retrospective clinical or pathophysiological investigations.
The method comparison studies showed a good agreement between the homogeneous assay and the precipitation technique in the control group. For example, there was a high correlation between both techniques (r = 0.90), and the regression line was very close to identity (slope = 0.96; intercept = 0.03 mmol/L). This correlation was somewhat lower than those reported in other evaluations; ranging between 0.95 and 0.99 (13)(14). Direct comparisons are not acceptable because these studies were performed on randomly obtained samples and the present study was conducted on samples from a control group of clinically healthy individuals who would not necessarily exhibit a wide range of HDL-C values. In addition, the Bland–Altman graphical assessment of agreement was good because there was no evidence of bias (the mean difference was very close to zero; -0.01 mmol/L) and the limits of agreement were narrow; both parameters indicate good concordance between the two methods of measurement.
With respect to samples from patients with certain pathologies,
however, a completely different situation prevailed. In the samples
from the elderly, ostensibly healthy subjects, the correlation between
both methods was still high (r = 0.95), but there was
an obvious inverse relationship between the differences and the average
of HDL-C concentrations (Fig. 3
, bottom right panel). In the samples
from patients who had evidence of premature atherosclerosis (AMI <55
years of age; group C) and the nephrotic syndrome patients (group D),
there was a lack of correlation between the two methods, as
demonstrated by the lower correlation coefficients as well as the
broader limits of agreement in Bland–Altman plots. The worst
concordance was observed in the samples from patients with liver
cirrhosis (group E). In these samples, the homogeneous method
underestimated HDL-C concentration, and the differences between the
methods were clearly dependent on the concentrations being measured:
values of up to -2.0 mmol/L when the average concentration being
measured was 2.5 mmol/L. Of considerable interest was that the
differences between the methods were related to serum bilirubin
and albumin concentrations; two biochemical markers indicative of liver
impairment.
This lack of agreement between the two methods in the samples from patients with certain pathologies is, perhaps, of crucial importance. The disagreement was within acceptable limits in the samples from the elderly group but progressively worsened in the samples of subjects with AMI and the nephrotic patients and produced very aberrant results in samples from patients with cirrhosis. The differences between the methods were not related to HDL-C or to the total cholesterol or triglyceride concentrations being measured; hence, a physiological explanation is difficult to propose. The only group in which an interpretation may be tentatively hypothesized is that of the cirrhotic patients. These subjects present with major alterations in HDL lipid and protein composition (27)(28), and perhaps more importantly, with structural alterations in the HDL particle, often exhibiting flat, discoidal shapes (termed "rouleaux") in the circulation and that tend to form aggregates in vitro (29). The possible interference of these alterations on the physico-chemical basis of the separation of the various lipoprotein classes on which the homogeneous HDL-C measurement depends is beyond the scope of the present investigation. However, the significant association between the method variation and the degree of liver dysfunction would lend credence to the suggestion that progressive physical alterations of the lipoproteins would produce progressive inaccuracy in the HDL-C measurement.
In conclusion, the present study demonstrates that, with respect to the parameters of method comparison, the direct method for measuring HDL-C is effective, inexpensive, and readily automated and lends itself to the screening of large populations. The limitations of the technique are highlighted in individual groups of patients in whom gross alterations in lipoproteins are encountered.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: HDL-C, high-density lipoprotein cholesterol; NCEP, National Cholesterol Education Program; CHOD-PAP, cholesterol oxidase-peroxidase; and AMI, acute myocardial infarction. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cyclodextrin. Clin Chem 1995;41:717-723.
The following articles in journals at HighWire Press have cited this article:
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  N. Ferre, J. Camps, J. Fernandez-Ballart, V. Arija, M. M. Murphy, S. Ceruelo, E. Biarnes, E. Vilella, M. Tous, and J. Joven Regulation of Serum Paraoxonase Activity by Genetic, Nutritional, and Lifestyle Factors in the General Population Clin. Chem., September 1, 2003; 49(9): 1491 - 1497. [Abstract] [Full Text] [PDF]
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M. M. Murphy, E. Vilella, S. Ceruelo, L. Figuera, M. Sanchez, J. Camps, G. Cuco, N. Ferre, A. Labad, N. Tasevska, et al. The MTHFR C677T, APOE, and PON55 Gene Polymorphisms Show Relevant Interactions with Cardiovascular Risk Factors Clin. Chem., February 1, 2002; 48(2): 372 - 375. [Full Text] [PDF]
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J. M. Simo, J. Camps, E. Vilella, F. Gomez, A. Paul, and J. Joven Instability of Lipoprotein(a) in Plasma Stored at -70 {degrees}C : Effects of Concentration, Apolipoprotein(a) Genotype, and Donor Cardiovascular Disease Clin. Chem., September 1, 2001; 47(9): 1673 - 1678. [Abstract] [Full Text] [PDF]
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F. Gomez, J. Camps, J. M. Simo, N. Ferre, and J. Joven Agreement Study of Methods Based on the Elimination Principle for the Measurement of LDL- and HDL-Cholesterol Compared with Ultracentrifugation in Patients with Liver Cirrhosis Clin. Chem., August 1, 2000; 46(8): 1188 - 1191. [Full Text] [PDF]
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P. Benlian, C. Cansier, G. Hennache, O. Khallouf, P. Bayer, F. Duron, F. Carrat, R. Couderc, O. Chazouilleres, J. Bardet, et al. Comparison of a New Method for the Direct and Simultaneous Assessment of LDL- and HDL-Cholesterol with Ultracentrifugation and Established Methods Clin. Chem., April 1, 2000; 46(4): 493 - 505. [Abstract] [Full Text] [PDF]
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A. Kondo, Y. Muranaka, I. Ohta, and T. Kanno Dynamic Reaction in a Homogeneous HDL-Cholesterol Assay Visualized by Electron Microscopy Clin. Chem., November 1, 1999; 45(11): 1974 - 1980. [Abstract] [Full Text] [PDF]
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J. Camps, J. M. Simo, S. Guaita, N. Ferre, and J. Joven Altered Composition of Lipoproteins in Liver Cirrhosis Compromises Three Homogeneous Methods for HDL-Cholesterol Clin. Chem., May 1, 1999; 45(5): 685 - 688. [Full Text] [PDF]
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