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Advantages and Disadvantages of Serum Cholesterol Determination by the

Kinetic vs the End Point Method

Article  in  American Journal of Clinical Pathology · July 2007

DOI: 10.1309/1ENNJCJRN682F1HP · Source: PubMed

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Clinical Chemistry / KINETIC AND END POINT CHOLESTEROL METHODS
Advantages and Disadvantages of Serum Cholesterol
Determination by the Kinetic vs the End Point Method
Pornpen Srisawasdi, PhD,1 Martin H. Kroll, MD,2 and Porntip H. Lolekha, MsC1
Key Words: Serum cholesterol; Kinetic method; End point method; Cholesterol oxidase; Enzymatic; Automated analyzer; Lipid;
Atherosclerosis; Coronary heart disease
DOI: 10.1309/1ENNJCJRN682F1HP
Abstract
Enzymatic assays for cholesterol determination can
use an end point or a kinetic method. We evaluated and
compared the performance of these methods. We
constructed user-defined methods on 3 automated
analyzers using Streptomyces cholesterol reagents to
evaluate the analytic performance of both methods.
Linearity (700-900 mg/dL) and stability of reagents (5-
11 weeks) depended on the analyzers. The coefficients
of variation for imprecision were 2.41% to 2.99% and
3.78% to 5.52% for the end point and kinetic methods,
respectively. The end point method showed less bias at
decision cut points (–0.8% to 1.3%) than did the kinetic
method (–1.1% to 3.6%) but was more affected by
interfering substances.
The advantages of the end point over the kinetic
method are better precision and lower reagent cost. The
end point imprecision fell within National Cholesterol
Education Program guidelines (≤3%), but the kinetic
did not. The kinetic method showed less interference
and required shorter analysis time.
906 Am J Clin Pathol 2007;127:906-918
906 DOI: 10.1309/1ENNJCJRN682F1HP
The National Cholesterol Education Program (NCEP)
has developed clinical guidelines for the diagnosis and treat-
ment of coronary disease.1 The guidelines offer cut points for
case finding and treatment based on the levels of serum total
cholesterol and high- and low-density lipoprotein. The accura-
cy of lipid and lipoprotein measurements is important. NCEP
expert panels have established requisite analytic performance
goals based on clinical needs for reliable patient classifica-
tion.2 The criteria for acceptable performance of cholesterol
analysis were bias (systematic error or overall accuracy) with-
in 3% of reference values and precision consistent with a coef-
ficient of variation (CV) of 3% or less. The total error goal for
cholesterol was set at 8.9% (% bias + 1.96 CV), requiring a
level of imprecision and bias.
Cholesterol may be quantitatively determined by chemical
or enzymatic methods. The current reference procedure is a
modification of the chemical method of Abell-Kendall using an
acetic anhydride–sulfuric acid reagent, even though limited by
interference, the use of corrosive reagents, and even small
quantities of moisture.3 Most clinical and most research mea-
surements of cholesterol use the enzymatic method.4 The enzy-
matic assay is specific, does not require corrosive chemicals,
and is easily adapted for automation. The assay is generally
performed as an end point5,6 or a kinetic7,8 method. Advantages
of the kinetic method over the end point method are shorter
analysis time, reduced effects of interfering substances, and
elimination of sample blank measurement.9,10 The main advan-
tage of the end point method over the kinetic method is its
insensitivity to minor changes in reaction conditions.11
According to previous studies,6,7 we found that
Streptomyces cholesterol oxidase was the best source of
enzyme for kinetic and end point cholesterol determinations.
© American Society for Clinical Pathology
 Clinical Chemistry / ORIGINAL ARTICLE

However, the optimal enzyme activity used in the reagent for
the end point method (200 U/L) was lower than for the kinet-
ic method (1,000 U/L). Using high enzyme activity for the
kinetic method may be affected by the addition of a competi-
tive inhibitor, 3,4-dichlorophenol, which could be used to ele-
vate the Michaelis constant of Streptomyces cholesterol oxi-
dase. To adapt this enzyme for clinical use, one must construct
user-defined kinetic and end point methods on the automated
analyzers. This study compares the usefulness of the kinetic
and end point assays for serum cholesterol determination.
3,4-dichlorophenol, which were obtained from BDH (Dorset,
England) and Aldrich Chemical (Milwaukee, WI), respectively.
Kinetic Cholesterol Reagent
To prepare the reagent, we dissolved Streptomyces cho-
lesterol oxidase, 1,000 U/L; cholesterol esterase from
Pseudomonas fluorescens, 400 U/L; peroxidase, 10,000 U/L;
sodium cholate, 3 mmol/L; 4-aminophenazone, 0.5 mmol/L;
phenol, 20 mmol/L; 3,4-dichlorophenol, 5 mmol/L; and Brij
35, 4.5 g/L, in phosphate buffer, 0.1 mol/L, pH 7.0.

Materials and Methods End Point Cholesterol Reagent

Equipment
We used 3 instruments, Vitalab Selectra Analyzer (E.
Merck, Darmstadt, Germany), Mega Analyzer (E. Merck),
and Dimension RxL (Dade Behring, Newark, DE) to assess
the kinetic and the end point cholesterol methods. The Vitalab
Selectra is an automated analyzer with small and medium
throughput (up to 180 tests per hour), and the Dimension RxL
and Mega are large analyzers (throughput, 500-800 tests per
hour). Moreover, the principle of photometry measurement is
different. Vitalab Selectra uses the single wavelength (505
nm), and the Mega (500 and 604 nm) and Dimension RxL
(510 and 600 nm) use dual wavelengths. The Mega and
Dimension RxL provide raw photometric data from the
bichromatic difference observed for photometric reading.
We prepared the reagent by dissolving Streptomyces choles-
terol oxidase, 200 U/L; cholesterol esterase from bovine pan-
creas, 200 U/L; peroxidase, 10,000 U/L; sodium cholate, 3
mmol/L; 4-aminophenazone, 0.5 mmol/L; phenol, 20 mmol/L;
and Triton X-100, 2 mL/L in phosphate buffer, 0.1 mol/L, pH 7.0.
Reference Samples
We used the serum-based calibrator (SMT, product No.
1.19720; E. Merck) with a cholesterol concentration of 240
mg/dL (6.21 mmol/L) to calibrate the methods implemented
on Vitalab Selectra and Mega. To calibrate cholesterol meth-
ods on the Dimension RxL, we used a set of CHOL
Calibrators (product No. REF DC 16, Dade Behring) consist-
ing of 3 cholesterol concentrations, 73, 240, and 425 mg/dL
(1.90, 6.20, and 11.00 mmol/L).

Reagents Procedure
All enzymes and chemicals were obtained from Sigma Details of the methods are given in ❚Table 1❚. We per-
Chemical (St Louis, MO), except 4-aminophenazone and formed the experimental cholesterol user-defined kinetic and

❚Table 1❚
Description of User-Defined Cholesterol Methods on Various Analyzers and the Dimension RxL Cholesterol Method*

User-Defined Parameters Dimension RxL

Vitalab Selectra Mega Dimension RxL Reference Assay

Kinetic End Point Kinetic End Point Kinetic End Point End Point

Reagent incubation time (min) 1.3 1.3 0.1 0.1 1.0 1.0 1.0
Delay (min) 2.5 — 4 — 2.5 — —
Incubation time (min) 4.5 11.5 3.0 9.0 2.0 10.0 6.3
Total time (min) 8.3 12.8 7.1 9.1 5.5 11.0 7.3
Wavelength (nm)
1 505 505 500 500 510 510 540
2 — — 604 604 600 600 452/700
Sample volume (µL) 3 3 3 3 3 3 3
Chase volume (H2O; µL) — — — — 20 20 60
Reagent volume (µL)
R1 300 300 300 300 300 300 88
R2 — — — — — — 26
Chase (H2O) — — — — 40 40 181
Principle of measurement Multi (linearity 1 10 1 3 1 1
(points) check)
No. of calibrator levels 1 1 1 1 3 3 3
* Dimension RxL, Dade Behring, Newark, DE; Mega and Vitalab Selectra, E. Merck, Darmstadt, Germany.

© American Society for Clinical Pathology Am J Clin Pathol 2007;127:906-918 907

907 DOI: 10.1309/1ENNJCJRN682F1HP 907
Srisawasdi et al / KINETIC AND END POINT CHOLESTEROL METHODS
end point methods on the Vitalab Selectra, Mega, and
Dimension RxL.
We used the Dimension RxL end point cholesterol
method (with cholesterol reagent product No. DF 27, Dade
Behring) as the reference comparison method. Our laboratory
was standardized through the Centers for Disease Control and
Prevention National Heart, Lung, and Blood Institute Lipid
Standardization Program; the bias and precision for choles-
terol measurement were within the acceptable limits (<3%).
Linearity and Reportable Range
To assess the linearity of each method, we created a set of
serum samples by admixing a high sample (without turbidity,
with a cholesterol level >900 mg/dL [23.27 mmol/L] as deter-
mined by the Dimension RxL) and the appropriate diluent (40
g/L of bovine albumin). All relative concentrations within this
set of serum samples were known to the experimenters.
Pooled serum was adjusted with 40 g/L of bovine serum albu-
min to obtain a cholesterol concentration of 900 mg/dL (23.27
mmol/L). A set of serum samples with cholesterol concentra-
tions ranging from 20 to 900 mg/dL (0.52-23.27 mmol/L) was
prepared accurately by proportionately mixing the pool with
40 g/L of bovine serum albumin, resulting in equally spaced
concentrations from 0 to 100 mg/dL (20-mg/dL spaced inter-
vals) and 100 to 900 mg/dL (100-mg/dL spaced intervals).
The absorbance values or the absorbance changes were plot-
ted against cholesterol concentrations. Linearity was evaluat-
ed by using the National Committee for Clinical Laboratory
Standards EP6-A guideline and the linear region reported as
the reportable range.12
Imprecision
We selected serum samples with low (~110 mg/dL [2.84
mmol/L]), middle (~190 mg/dL [4.91 mmol/L]), and high
(~400 mg/dL [10.34 mmol/L]) cholesterol concentrations.
The within-run (20 replicates in the same run) and between-
run (20 consecutive days) imprecision values were determined
in each serum sample. The means, SDs, and CVs (CV =
SD/mean × 100%) were calculated and tabulated.
Recovery
We performed the recovery study by mixing serum sam-
ples containing low, middle, and high cholesterol concentra-
tions in a 1:1 ratio (low + middle, low + high, and middle +
high). We calculated the recovery of cholesterol from the test
methods compared with the Dimension RxL method in per-
centages.
Comparison
We compared test method cholesterol levels in 100 serum
samples from hospitalized patients with those from the refer-
ence end point method (Dimension RxL cholesterol assay).
908 Am J Clin Pathol 2007;127:906-918
908 DOI: 10.1309/1ENNJCJRN682F1HP
The regression equations, correlation coefficients, bias, and
the SD of the residuals (Sy/x) values obtained between the
methods were calculated. We also used the difference plot to
examine agreement between 2 methods. The 95% confidence
interval for the mean difference was calculated.
Interference Study
We prepared the sets of hemolyzed (hemoglobin, 0-15.0
g/L), icteric (bilirubin, 0-80 mg/dL), and turbid (absorbance at
670 nm, 0-2.334) samples. A 1:2 dilution of each interference
sample with pooled serum of low, middle, and high choles-
terol levels was made to obtain 3 sets of various degrees of
interfering substances. The cholesterol level in each sample’s
set was determined by using the cholesterol reagents.
Stability
The stability of each cholesterol reagent was studied by
analyzing the middle (180 mg/dL [4.65 mmol/L]) and high
(340 mg/dL [8.79 mmol/L]) cholesterol samples for 3 months.
The reagents were stored in a refrigerator (2°C-8°C).
Statistical Methods
Mean, SD, CV (%), and the correlation and regression
analyses were performed with Microsoft Excel, Microsoft,
Redmond, WA. Bias error was calculated from the regression
analysis at cholesterol decision cut points of 200 and 240
mg/dL (5.17 and 6.21 mmol/L, respectively). Random error
was obtained from the CV percentage based on the between-
run imprecision study. We used the F test to evaluate the preci-
sion between the kinetic and end point methods. Results were
considered statistically significant when P was less than .05.
Results
Linearity
❚Figure 1❚ shows the linearity of serum cholesterol values
obtained from the kinetic and the end point assays on the
Vitalab Selectra, Mega, and Dimension RxL. The upper end of
the reportable range for the kinetic assays using Dimension
RxL (700 mg/dL [18.10 mmol/L]) was lower than from the
Vitalab Selectra (800 mg/dL [20.69 mmol/L]) and the Mega
(900 mg/dL [23.27 mmol/L]). With the end point method, the
upper end linearity of the Dimension RxL (900 mg/dL [23.27
mmol/L]) was higher than with the Vitalab Selectra (800 mg/dL
[20.69 mmol/L]) and the Mega (700 mg/dL [18.10 mmol/L]).
Reproducibility
❚Table 2❚ shows the within- and between-run precision
obtained from serum samples with low, middle, and high cho-
lesterol concentrations. The range of the average within-run
© American Society for Clinical Pathology
 Clinical Chemistry / ORIGINAL ARTICLE

A B
0.12 1.8
Absorbance Change at 505 nm

1.6

Absorbance at 505 nm
0.10
1.4
0.08 1.2
1.0
0.06
0.8
0.04 0.6
0.4
0.02
0.2
0.00 0.0
0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 800 900
Serum Cholesterol (mg/dL) Serum Cholesterol (mg/dL)

C D
0.12 1.8
Absorbance Change at 500 nm

1.6
0.10

Absorbance at 500 nm
1.4
0.08 1.2
1.0
0.06
0.8
0.04 0.6
0.4
0.02
0.2
0.00 0.0
0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 800 900
Serum Cholesterol (mg/dL) Serum Cholesterol (mg/dL)

E F
0.05
Absorbance Unit Change at 510 nm

0.50
0.45
Absorbance Unit at 510 nm

0.04 0.40
0.35
0.03 0.30
0.25
0.02 0.20
0.15
0.01 0.10
0.05
0.00 0.00
0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 800 900
Serum Cholesterol (mg/dL) Serum Cholesterol (mg/dL)

❚Figure 1❚ Linearity of serum cholesterol values obtained from the kinetic cholesterol assay (A, C, and E) and the end point
assay (B, D, and F) using the Vitalab Selectra (A and B, E. Merck, Darmstadt, Germany), Mega (C and D, E. Merck), and
Dimension RxL (E and F, Dade Behring, Newark, DE) analyzers. Absorbance change is the absorbance change per minute;
absorbance, the final absorbance; absorbance unit change, the milliabsorbance unit change per minute; and absorbance unit, the
final milliabsorbance unit. Cholesterol values are given in conventional units; to convert to Système International units (mmol/L),
multiply by 0.02586.

© American Society for Clinical Pathology Am J Clin Pathol 2007;127:906-918 909

909 DOI: 10.1309/1ENNJCJRN682F1HP 909
Srisawasdi et al / KINETIC AND END POINT CHOLESTEROL METHODS

imprecision for the kinetic method was 1.82% to 2.49% and ❚Table 2❚
for the end point method, 1.44% to 2.45%. For between-run Within- and Between-Run Imprecision of Total Serum
Cholesterol Values Using Kinetic and End Point Methods on
imprecision, the end point method (CV, 2.41%-2.99%) was Various Analyzers*
less than the kinetic method (CV, 3.78%-5.52%). The
observed differences between the kinetic and end point meth- Coefficient of Variation (%)

ods were statistically significant (P < .05) for between-run Within-Run (n = 20) Between-Run (n = 20)
imprecision but not for within-run imprecision.
Kinetic End Point Kinetic End Point

Accuracy Vitalab Selectra

Low 2.88 2.15 5.70 2.78
The analytic recovery of serum cholesterol obtained from Middle 1.71 1.20 5.80 2.44
the implemented methods is given in ❚Table 3❚. The averages High 1.44 0.96 5.04 2.56
ranged from 97.3% to 102.1% and 95.7% to 103.4% for the Average 2.01 1.44 5.52 2.59
Mega
kinetic and end point determinations, respectively. Low 4.13 3.49 5.08 3.30
We compared cholesterol measured with the test methods Middle 2.23 1.47 4.46 2.70
High 1.11 2.39 3.75 2.98
with the reference end point method by least-square linear Average 2.49 2.45 4.43 2.99
regression analysis ❚Figure 2❚. The regression lines were very Dimension RxL
Low 2.01 2.28 4.88 2.46
close to the identity line with correlation coefficients greater Middle 1.56 1.36 3.18 2.44
than 0.99. The ranges in values for the 95% confidence inter- High 1.88 1.22 3.27 2.32
Average 1.82 1.62 3.78 2.41
val of slope and y-intercept were very tight ❚Table 4❚.
To better illustrate the comparative performance of the * For proprietary information, see Table 1.
implemented assays, the results from the same patient serum
samples were compared separately with the Dimension RxL
method. The bias plots (difference between the test method mmol/L), and 7.2 mg/dL (0.19 mmol/L) and –16.2 to 12.7
and the Dimension RxL method plotted vs the Dimension mg/dL (–0.42 to 0.33 mmol/L) for the kinetic method by
RxL results) are shown in ❚Figure 3❚. The kinetic methods Vitalab Selectra, Mega, and Dimension RxL, respectively. For
reported mean biases of 8.25 mg/dL, 3.95, and –1.75 mg/dL the end point method, they were 7.0 mg/dL (0.18 mmol/L)
(0.21, 0.10, and –0.05 mmol/L, respectively) using the Vitalab and –13.1 to 14.7 mg/dL (–0.34 to 0.38 mmol/L), 7.3 mg/dL
Selectra, Mega, and Dimension RxL, respectively, and the end (0.19 mmol/L) and –15.8 to 14.7 mg/dL (–0.41 to 0.38
point method reported mean biases of 0.89, –1.24, and 3.58 mmol/L), and 5.4 mg/dL (0.14 mmol/L) and –7.3 to 14.3
mg/dL (0.02, –0.03, and 0.09 mmol/L, respectively). mg/dL (–0.19 to 0.37 mmol/L), respectively.
The Vitalab Selectra and Mega kinetic and end point
methods showed a constant slope compared with the commer- Analytic Error of the Implemented Methods
cial Dimension RxL method. The 95% confidence intervals of We estimated the bias error as a percentage of each cho-
the differences were slightly larger for the kinetic methods. lesterol assay at the clinical decision cut points of serum cho-
The SD of differences and the 95% confidence limits of dif- lesterol concentrations (200 and 240 mg/dL [5.17 and 6.21
ferences (mean bias ± 2 SD) were 9.0 mg/dL (0.23 mmol/L) mmol/L, respectively]) from the regression analysis in ❚Table
and –9.7 to 25.9 mg/dL (–0.25 to 0.67 mmol/L), 11.1 mg/dL 5❚. The systematic errors of all cholesterol methods compared
(0.29 mmol/L) and –18.1 to 26.3 mg/dL (–0.47 to 0.68 with the Dimension RxL method ranged from –1.1% to 3.6%

❚Table 3❚
Recovery of Total Serum Cholesterol Obtained From the Kinetic and End Point Methods on Various Analyzers*

Observed Value (mg/dL) Recovery (%)

Expected Vitalab Selectra Mega Dimension RxL Vitalab Selectra Mega Dimension RxL
Value
(mg/dL) M1 M2 M1 M2 M1 M2 M1 M2 M1 M2 M1 M2

L + M (1:1) 147 140 135 145 150 150 151 95.2 91.8 98.6 102.0 102.0 102.7
L + H (1:1) 229 225 224 227 241 233 239 98.3 97.8 99.1 105.2 101.7 104.4
M + H (1:1) 273 269 266 272 281 280 278 98.5 97.4 99.6 102.9 102.6 101.8
Average 97.3 95.7 99.1 103.4 102.1 103.0
* M1, kinetic method; M2, end point method. Cholesterol concentrations: low (L), 103 mg/dL (2.66 mmol/L; middle (M), 190 mg/dL (4.91 mmol/L); high (H), 355 mg/dL (9.18
mmol/L). For proprietary information, see Table 1.

910 Am J Clin Pathol 2007;127:906-918 © American Society for Clinical Pathology

910 DOI: 10.1309/1ENNJCJRN682F1HP
 Clinical Chemistry / ORIGINAL ARTICLE

A B
600 600

Vitalab Selectra (mg/dL)

Vitalab Selectra (mg/dL)

500 500

400 400

300 300

200 200

100 100

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

C D
600 600

500 500

Mega (mg/dL)
Mega (mg/dL)

400 400

300 300

200 200

100 100

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

E F
600 600

500 500
Dimension RxL (mg/dL)

Dimension RxL (mg/dL)

400 400

300 300

200 200

100 100

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

❚Figure 2❚ Correlation of total serum cholesterol values determined from the kinetic cholesterol assay (A, C, and E) and the end
point assay (B, D, and F) using the Vitalab Selectra (A and B), Mega (C and D), and Dimension RxL (E and F) analyzers with the
commercial Dimension RxL cholesterol method. A, y = 1.06x – 5.68; r = 0.996; bias = 8.25; Sy/x = 7.439. B, y = 1.06x – 11.64; r
= 0.998; bias = 0.89; Sy/x = 5.278. C, y = 1.11x – 19.98; r = 0.997; bias = 3.95; Sy/x = 6.974. D, y = 1.04x – 9.74; r = 0.997; bias
= –1.24; Sy/x = 6.67. E, y = 0.97x + 4.63; r = 0.996; bias = –1.75; Sy/x = 6.989. F, y = 1.00x + 2.65; r = 0.998; bias = 3.58; Sy/x =
5.498. Cholesterol values are given in conventional units; to convert to Système International units (mmol/L), multiply by
0.02586. For proprietary information, see Figure 1.

© American Society for Clinical Pathology Am J Clin Pathol 2007;127:906-918 911

911 DOI: 10.1309/1ENNJCJRN682F1HP 911
Srisawasdi et al / KINETIC AND END POINT CHOLESTEROL METHODS

A B
Kinetic Test – Dimension RxL (mg/dL) 50 50

End Point – Dimension RxL (mg/dL)

40 40
30 30
20 20
10 10
0 0
–10 –10
–20 –20
–30 –30
0 100 200 300 400 500 0 100 200 300 400 500
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

C D
Kinetic Test – Dimension RxL (mg/dL)

50 50

End Point – Dimension RxL (mg/dL)

40 40
30 30
20 20
10 10
0 0
–10 –10
–20 –20
–30 –30
0 100 200 300 400 500 0 100 200 300 400 500
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

E F
50
End Point – Dimension RxL (mg/dL)
Kinetic Test – Dimension RxL (mg/dL)

50

40 40

30 30

20 20

10 10

0 0

–10 –10

–20 –20

–30 –30
0 100 200 300 400 500 0 100 200 300 400 500
Dimension RxL (mg/dL) Dimension RxL (mg/dL)

❚Figure 3❚ Difference plots of serum cholesterol values obtained from the kinetic cholesterol assay (A, C, and E) and the end
point assay (B, D, and F) using the Vitalab Selectra (A and B), Mega (C and D), and Dimension RxL (E and F) analyzers compared
with the commercial Dimension RxL cholesterol assay. The y-axis is the bias of cholesterol values obtained between the
implemented methods and the Dimension RxL cholesterol assay, and the x-axis is the cholesterol concentration obtained from
the commercial method. The solid line is the mean difference plot between 2 methods. The dotted lines represent 95%
confidence limits of the differences (mean bias ± 2 SD). Bias (mg/dL) and SD of difference (mg/dL), respectively: A, 8.25 and 9.0;
B, 0.89 and 7.0; C, 3.95 and 11.1; D, –1.24 and 7.3; E, –1.75 and 7.2; F, 3.58 and 5.4. Cholesterol values are given in conventional
units; to convert to Système International units (mmol/L), multiply by 0.02586. For proprietary information, see Figure 1.

912 Am J Clin Pathol 2007;127:906-918 © American Society for Clinical Pathology

912 DOI: 10.1309/1ENNJCJRN682F1HP
 Clinical Chemistry / ORIGINAL ARTICLE

❚Table 4❚
Correlation of Implemented Methods on Various Instruments and the Reference Assay*

Coefficients (95% CI)

Method Mean (mg/dL) r SE of Estimate Bias (mg/dL) Slope Intercept

Vitalab Selectra
Kinetic 226 0.996 7.44 8.25 1.06 (1.05 to 1.08) –5.68 (–10.0 to –1.35)
End point 219 0.998 5.28 0.89 1.06 (1.04 to 1.07) –11.64 (–14.7 to –8.57)
Mega
Kinetic 222 0.997 6.97 3.95 1.11 (1.09 to 1.13) –19.98 (–24.1 to –15.9)
End point 217 0.997 6.67 –1.24 1.04 (1.02 to 1.06) –9.74 (–13.6 to –5.83)
Dimension RxL
Kinetic 216 0.996 6.99 –1.75 0.97 (0.95 to 0.99) 4.63 (0.54 to 8.73)
End point 221 0.998 5.50 3.58 1.00 (0.99 to 1.02) 2.65 (–0.54 to 5.87)
Commercial 218 — — — — —

CI, confidence interval.

* The reference method was the Dimension RxL. For proprietary information, see Table 1. Values are given in conventional units; to convert to Système International units

(mmol/L), multiply by 0.02586.

and –0.8% to 1.3% for the kinetic and end point methods, ❚Table 5❚
Analytic Error of Implemented Cholesterol Assays*
respectively. The systematic errors of all end point methods
were within the allowable bias recommended by the NCEP Bias Error (%)†
guidelines for cholesterol, less than 3% at decision levels,2 but
200 240 Imprecision (%)‡
those of the kinetic method using the Vitalab Selectra (3.1%-
3.6%) were higher. The means and SDs of the cholesterol val- Vitalab Selectra
Kinetic method 3.1 3.6 5.5
ues calculated from each linear regression at the cut point val- End point method 0.2 1.2 2.6
ues of 200 and 240 mg/dL (5.17 and 6.21 mmol/L, respective- Mega
Kinetic method 1.0 2.6 4.4
ly) were 203 ± 3.1 mg/dL (5.25 ± 0.08 mmol/L) and 244 ± 4.6 End point method –0.8 0.0 3.0
mg/dL (6.31 ± 0.12 mmol/L), respectively, for the kinetic Dimension RxL
method and 201 ± 1.9 mg/dL (5.20 ± 0.05 mmol/L) and 242 Kinetic method –0.7 –1.1 3.8
End point method 1.3 1.1 2.4
± 1.2 mg/dL (6.25 ± 0.03 mmol/L), respectively, for the end
* For proprietary information, see Table 1.
point method. † Calculated at the clinical decision cut point of serum cholesterol (200 and 240
mg/dL [5.17 and 6.21 mmol/L, respectively]) from the regression equations.
‡ Calculated from 20 consecutive day measurements of the implemented methods.
Interference
The pools were the same as in Table 2.
The interfering effects from hemoglobin, bilirubin, and
turbidity on the total cholesterol assay results are given in
❚Table 6❚. Interfering substances produce significant effects Reagent Stability
when the results differ by more than 10% (higher or lower) The stability of reagents for the kinetic and end point cho-
from the original cholesterol concentration.13 Hemoglobin lesterol determinations depended on the analyzer ❚Figure 4❚.
at a concentration of 0.75 g/dL (7.5 g/L) only increased The reagent stability used in the kinetic method by the Mega
serum cholesterol recovery on the end point measurement (11 weeks) was longer than for the Vitalab Selectra (8 weeks)
using the Vitalab Selectra. The degrees of turbidity of 4+ and Dimension RxL (5 weeks). For the end point assay, the
(absorbance, >1.167 at 670 nm) and 2+ (absorbance, stability with the Mega (6 weeks) was shorter than with the
>0.778 at 670 nm) gave a positive bias on serum choles- Vitalab Selectra (10 weeks) and Dimension RxL (11 weeks).
terol values with the kinetic method on the Mega and
Dimension RxL, respectively, whereas a negative bias was
found with the method on the Vitalab Selectra. With the end
Discussion
point method, the degree of turbidity at 1+ (absorbance,
>0.389 at 670 nm) resulted in increased serum cholesterol The enzymatic method for cholesterol determination may
values with all analyzers. Bilirubin concentrations greater use an end point or a kinetic method. The analytic reactions of
than 10 mg/dL (171.0 µmol/L) imparted a negative bias for the enzymes cholesterol esterase, cholesterol oxidase, and per-
all cholesterol determinations except the end point method oxidase convert 4-aminophenazone and a phenol to a colored
with the Vitalab Selectra, which started at a concentration of quinoneimine dye after the oxidation of cholesterol.
20 mg/dL (342 µmol/L). Streptomyces provides a good source of cholesterol oxidase in

© American Society for Clinical Pathology Am J Clin Pathol 2007;127:906-918 913

913 DOI: 10.1309/1ENNJCJRN682F1HP 913
Srisawasdi et al / KINETIC AND END POINT CHOLESTEROL METHODS

❚Table 6❚
Interference Study on Serum Cholesterol Determination*

Cholesterol Concentration (mg/dL)

Kinetic Method End Point Method

Degree Interferent Vitalab Selectra Mega Dimension RxL Vitalab Selectra Mega Dimension RxL

Hemolysis Hemoglobin (g/L)

Baseline 0 69 71 81 68 66 83
± 0.1 70 71 80 68 65 82
1+ 0.9 68 70 82 70 67 83
2+ 1.9 71 72 81 71 69 84
3+ 3.8 73 70 81 76 70 87
4+ 7.5 73 69 81 85† 75 89
Baseline 0 187 190 178 183 179 189
± 0.1 188 198 174 181 179 185
1+ 0.9 188 188 174 182 181 187
2+ 1.9 189 190 177 183 180 188
3+ 3.8 192 191 176 189 183 188
4+ 7.5 191 186 174 196† 184 190
Baseline 0 285 278 254 271 271 269
± 0.1 285 271 251 269 270 269
1+ 0.9 287 276 254 271 267 274
2+ 1.9 286 271 257 274 267 271
3+ 3.8 284 278 247 274 271 268
4+ 7.5 287 277 245 282† 272 271
Turbidity Absorbance (670 nm)
Baseline 0 70 71 81 68 66 83
± 0.195 68 74 88 76 72 89
1+ 0.389 63 76 91 83† 79† 95†
2+ 0.778 51‡ 80 100† 98 90 104
3+ 0.973 43 80 105 103 95 110
4+ 1.167 39 83† 106 114 103 115
Baseline 0 187 190 180 183 179 189
± 0.195 179 193 189 191 188 204
1+ 0.389 180 194 189 197† 195† 207†
2+ 0.778 164‡ 197 200† 212 207 214
3+ 0.973 162 200 202 222 214 217
4+ 1.167 158 201† 204 229 224 225
Baseline 0 285 278 254 271 271 269
± 0.195 281 283 262 279 281 280
1+ 0.389 279 288 268 287† 285† 288†
2+ 0.778 274‡ 286 264† 316 295 302
3+ 0.973 256 299 276 313 296 304
4+ 1.167 254 300† 278 320 311 308
Icteric Bilirubin (mg/dL)
Baseline 0 68 69 83 69 64 84
± 5.0 68 60 81 64 59 75
1+ 10.0 60‡ 42‡ 67‡ 63 52‡ 73‡
2+ 20.0 34 3 39 59‡ 44 62
3+ 30.0 8 –37 15 58 39 57
4+ 40.0 –7 –71 –3 58 38 52
Baseline 0 188 189 192 180 180 192
± 5.0 181 181 181 177 170 185
1+ 10.0 159‡ 176‡ 173‡ 171 160‡ 172‡
2+ 20.0 144 158 154 164‡ 142 153
3+ 30.0 112 127 119 153 124 136
4+ 40.0 89 88 91 146 107 120
Baseline 0 277 281 251 269 265 271
± 5.0 275 275 251 264 264 257
1+ 10.0 264‡ 265‡ 253‡ 258 244‡ 253‡
2+ 20.0 221 255 245 249‡ 230 230
3+ 30.0 180 247 222 235 202 214
4+ 40.0 171 224 210 226 192 198
* Hemoglobin values are given in Système International (SI) units; to convert to conventional units (g/dL), divide by 10.0; bilirubin values are given in conventional units; to
convert to SI units (µmol/L), multiply by 17.1. Bold data indicate cholesterol values with significant interference. For proprietary information, see Table 1.
† Starting significant positive interference (>10% increasing from baseline level).13
‡ Starting significant negative interference (>10% decreasing from baseline level).

914 Am J Clin Pathol 2007;127:906-918 © American Society for Clinical Pathology

914 DOI: 10.1309/1ENNJCJRN682F1HP
 Clinical Chemistry / ORIGINAL ARTICLE

A Absorbance Change at 505 nm B

0.05 0.7

Absorbance at 505 nm
0.6
0.04
0.5
0.03 0.4

0.02 0.3
0.2
0.01
0.1
0.00 0.0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Storage Time (wk) Storage Time (wk)

C D
Absorbance Change at 500 nm

0.05 0.7

Absorbance at 500 nm
0.6
0.04
0.5
0.03
0.4
0.02 0.3
0.2
0.01
0.1
0.00 0.0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Storage Time (wk) Storage Time (wk)

E F
Absorbance Unit Change at 510 nm

0.020 0.25
Absorbance Unit at 510 nm

0.20
0.015

0.15
0.010
0.10

0.005
0.05

0.000 0.00
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Storage Time (wk) Storage Time (wk)

❚Figure 4❚ Stability of the cholesterol reagent containing cholesterol oxidase from Streptomyces for the kinetic (A, C, and E) and
end point (B, D, and F) methods kept at 2°C-8°C using the Vitalab Selectra (A and B), Mega (C and D), and Dimension RxL (E
and F) analyzers. Diamonds, normal cholesterol values (180 mg/dL); squares, high cholesterol values (340 mg/dL). Absorbance
change is the absorbance change per minute; absorbance, the final absorbance; absorbance unit change, the milliabsorbance
unit change per minute; and absorbance unit, the final milliabsorbance unit. Dotted lines represent the assumed line of the
stable reagent. Cholesterol values are given in conventional units; to convert to Système International units (mmol/L), multiply by
0.02586. For proprietary information, see Figure 1.

© American Society for Clinical Pathology Am J Clin Pathol 2007;127:906-918 915

915 DOI: 10.1309/1ENNJCJRN682F1HP 915
Srisawasdi et al / KINETIC AND END POINT CHOLESTEROL METHODS
serum cholesterol determinations by the end point and kinetic
assays.6,7 To compare the advantages and disadvantages of the
end point and kinetic methods, we developed user-defined
methods using the Vitalab Selectra, Mega, and Dimension
RxL analyzers. The specifications are listed in Table 1.
The upper reportable range of cholesterol obtained from
the end point and kinetic assays ranged from 700 to 900
mg/dL (18.10-23.27 mmol/L), depending on the analyzer and
the type of measurement. The reagents maintained these
reportable ranges for the shelf life of the reagents (5-11
weeks). An upper reportable range of 700 mg/dL (18.10
mmol/L) is sufficient for most clinical uses.
The kinetic method recovered cholesterol equally as well
as the end point method, as is apparent in Table 3. The results
of linear regression showed good association between the
kinetic and end point methods and the Dimension RxL choles-
terol method (reference method). Bias plots of the Vitalab
Selectra and Mega revealed small proportional biases depend-
ent on the cholesterol concentration (Figure 3). Assays on the
Dimension RxL did not demonstrate such biases. Such results
may be due to the assigned value of the calibrator used for
each cholesterol method. The Vitalab Selectra and Mega
methods used a single concentration of calibrator, whereas the
Dimension RxL methods used 3 concentrations of calibrator
(multipoint calibration) (Table 1) covering the range of 73 to
425 mg/dL. Thus, using multipoint calibration may be superi-
or to single-point calibration. On the other hand, the biases
observed for the Vitalab Selectra and Mega analyzers may be
greater than the biases for the Dimension RxL because of dif-
ferences between analyzers rather than between methods.
One could correct the observed biases for the kinetic
method as seen with these 2 analyzers by reassigning the
calibrator set points. The calibrator used for the Vitalab
Selectra and Mega kinetic methods in this study was original-
ly designed for use with an end point commercial method at
a concentration of 240 mg/dL. Because the analytic concen-
trations of calibrators often include specific biases, intro-
duced to force them to work with specific analyzers, one
would not expect the values of calibrator assigned for the
end point method to behave in an unbiased manner for the
kinetic method.
The between-run precision results obtained from the end
point methods varied significantly from those of the kinetic
method (P < .05), whereas the within-run precision varied lit-
tle. The end point method showed good precision within the
analytic criteria (CV, <3.0%) recommended by NCEP.2 The
end point method is less sensitive to minor changes in reaction
conditions, whereas the kinetic method requires strict equiva-
lence of enzyme activity in each reaction mixture and tightly
controlled pH and temperature. More frequent calibration,
perhaps as frequent as once a day, may compensate for these
sensitivities and improve precision of the kinetic method.
916 Am J Clin Pathol 2007;127:906-918
916 DOI: 10.1309/1ENNJCJRN682F1HP
The kinetic method on the Dimension RxL demonstrated
better precision than on the Vitalab Selectra and Mega. The
95% confidence limits of the differences (Figure 3) were
slightly larger for the Vitalab Selectra and Mega kinetic method
than for the Dimension RxL. The type of reaction cuvette may
have caused these differences. The Dimension RxL (Dade
Behring) uses disposable cuvettes, whereas the Vitalab Selectra
and Mega methods employ reusable cuvettes. Carryover from
one sample to the next may have caused contamination.
To achieve reliable classification of patients, NCEP
expert laboratory panels2 have established requisite analytic
performance goals based on clinical needs. The NCEP recom-
mends that the total error (<9%) can be achieved by an impre-
cision of 3% or less and a bias compared with the reference
method of 3% or less. Our data show that the imprecision and
the bias criterion were met by all end point methods (Table 5)
and, thus, have met the NCEP total error requirements. On the
other hand, the imprecision errors of all kinetic methods were
higher than those of the end point methods. Moreover, the
kinetic method on the Vitalab Selectra demonstrated a bias of
more than 3%. The total error of the kinetic methods exceed-
ed the NCEP requirements. The kinetic method on the
Dimension RxL differed from the other analyzers (Table 5 and
Figure 3); its mean analytic error was low and within the sys-
tematic error requirement. If its between-run precision could
be reduced, it might meet the NCEP total error criteria.
Addition of free hemoglobin did not interfere with deter-
mination of serum cholesterol values by kinetic and end point
methods using 2 wavelength measurements (Dimension RxL
and Mega, Table 6). But hemoglobin interfered with the
Vitalab Selectra, which uses only 1 wavelength. These results
suggest that clinical laboratory instruments using only 1 wave-
length (small automated analyzers or point-of-care devices)
are more prone to hemoglobin interference when using the
end point method. The kinetic cholesterol assay was free of
hemoglobin interference, which suggests the kinetic method
may be superior to the end point method in small analyzers
and, especially, point-of-care devices, where the chance of
hemolysis is greater owing to skin puncture and/or undiluted
samples. Also, when whole blood samples are used, the oper-
ator is never aware when hemolysis has occurred.
Turbidity interfered less with the kinetic than with the
end point method. For the kinetic method, the multiple
measurements subtract out the light-scattering effects of
lipid particles and, thus, eliminate the turbidity interfer-
ence.14 Even though the Dimension RxL and the Mega ana-
lyzers use bichromatic wavelength blanking, turbidity still
caused a significant positive interference with these meth-
ods. Because lipid particles scatter more light at 500 nm than
at 600 nm and the wavelength for blanking is set near 600
nm, the blank fails to adequately compensate for the degree
of lipid scattering caused by turbidity.15
© American Society for Clinical Pathology

Only the kinetic method on the Vitalab Selectra analyzer
had negative interference caused by turbidity. All other meth-
ods had positive interference. The Vitalab Selectra differs
from the other analyzers because it uses only 1 wavelength.
On adding a turbid sample to the reagents, the initial
absorbance rose to a high value, but the presence of detergent
(Brij 35) and Pseudomonas fluorescens cholesterol esterase
slowly diminished the turbidity. We suggest that the decreased
light scattering (detected as absorbance) over time subtracts
absorbance from the main reaction, decreasing the value of the
slope and altering the absorbance rate.
All cholesterol assays showed negative interference by
bilirubin (Table 6). Spectral and chemical mechanisms explain
bilirubin interference with enzymatic peroxidase reactions.16
For the end point method, spectral interference producing a
false-positive result might cancel the false-negative result pro-
duced by chemical interference. Our data show that bilirubin
caused negative interference with all of the methods, suggest-
ing that chemical interference predominates.
Although the kinetic method can eliminate absorbance
effects of bilirubin interference at 505 nm, it cannot eliminate
bilirubin’s competition with the color reaction for hydrogen
peroxide.14 Many procedures have been suggested for the cor-
rection of bilirubin interference. Addition of potassium ferro-
cyanide to the reagent for end point methods14,16 removes the
contribution of bilirubin to the chemical reaction. The addition
of potassium ferrocyanide to the cholesterol reagent may suc-
cessfully eliminate bilirubin interference with the enzymatic
kinetic method as well.
Most enzymatic methods for cholesterol determination
use the end point method. At present, commercial kinetic
enzymatic methods have been developed for the determina-
tion of glucose (Dade Behring), urea (Dade Behring), and
triglycerides (Dade Behring). However, most commercial
cholesterol assays still use the end point method. Our study
provides some insight into the popularity of these end point
cholesterol assays. End point methods have better precision
and lower reagent costs than the kinetic ones. The imprecision
of the end point method was within NCEP guidelines, where-
as imprecision of the kinetic methods was not. The cost of the
enzyme used in the reagent for the end point method
($0.16/mL) is less than for that used in the kinetic method
($0.81/mL).
A major disadvantage of the end point method is sensitivi-
ty to interfering substances. One frequently finds hyperlipide-
mic samples with all types of patients. Centrifugation at 12,000g
for at least 10 minutes may separate serum or plasma lipids by
flotation and is used for many analytic methods but cannot be
used for cholesterol determination. We believe that a kinetic
method may be preferred when dealing with turbid samples.
Kinetic methods require shorter analysis time. The
kinetic method might prove useful in analyzers when a short
© American Society for Clinical Pathology
Clinical Chemistry / ORIGINAL ARTICLE
measurement time is desired, as in point-of-care testing, in
which results could be displayed within 5 minutes after spec-
imen application.
Because hemolysis occurs frequently, and in point-of-
care testing it would be unobserved, kinetic methods would be
preferred over end point methods. Also, because hyperlipi-
demia occurs frequently and would not be observed in the
point-of-care setting, kinetic methods would be preferred.
Even though bilirubin at high concentrations showed more
interference with the kinetic than with the end point method,
cholesterol values in such samples have little significance for
cardiac risk assessment (the patient is at much greater risk for
complications of liver disease) or other purpose, and one
would not expect to use point-of-care testing for such patients.
Furthermore, one could easily identify such patients by
observing jaundice. Thus, even though as currently formulat-
ed end point methods seem superior to kinetic methods for
central laboratory determination, kinetic methods may be
superior to end point methods for point-of-care testing.
Conclusion
Cholesterol reagents containing Streptomyces cholesterol
oxidase work for the end point and kinetic methods for cho-
lesterol determination. The end point method seems superior
to the kinetic method because of better accuracy (analytic
bias, <3%) and precision (<3%). Nevertheless, the kinetic
method demonstrates several advantages over the end point
method in its lower sensitivity to interfering substances and
shorter analysis times. This method could be useful, especial-
ly in point-of-care testing.
From the 1Division of Clinical Chemistry, Department of
Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol
University, Bangkok, Thailand; and 2Department of Pathology and
Laboratory Medicine, Dallas Veterans Affairs Medical Center and
University of Texas Southwestern Medical School, Dallas.
Supported in part by the Thai Research Fund Royal Golden
Jubilee PhD program, Bangkok, Thailand.
Address reprint requests to Dr Srisawasdi: Division of Clinical
Chemistry, Dept of Pathology, Faculty of Medicine Ramathibodi
Hospital, Rama VI Road, Rajthevee, Bangkok 10400, Thailand.
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© American Society for Clinical Pathology