1.0 INTENDED USE
For the direct quantitative
determination of low-density lipoprotein cholesterol (LDL-C) in human serum or
plasma. For In Vitro diagnostic use
only.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Plasma lipoproteins are
spherical particles containing varying amounts of cholesterol, triglycerides,
phospholipids and proteins. The phospholipid, free cholesterol and protein
constitute the outer surface of the lipoprotein particle, the inner core contains
mostly esterified cholesterol and triglycerides. These particles serve to
solubilize and transport cholesterol and triglycerides in the bloodstream.
The relative proportions of
protein and lipid determine the density of these plasma lipoproteins and
provide a basis on which to begin their classification (11.1). These classes
are: very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and
high-density lipoprotein (HDL). Numerous clinical studies have shown that the
different lipoprotein classes have varied effects. (11.2-11.4). The studies all
point to LDL cholesterol as the key factor in the pathogenesis of
atherosclerosis and coronary artery disease (CAD) (11.2-11..8), while HDL
cholesterol has often been observed to have a protective effect. Even within the normal range of total
cholesterol concentrations, an increase in LDL cholesterol can occur with an
associated increased risk for CAD (11.4).
Over the years a variety of
methods have been employed for the determination, or estimation, of LDL
cholesterol. The Friedewald equation, in
a variety of forms, has been most frequently used for the estimation of LDL
cholesterol. However, its usefulness is
limited and its accuracy has been questioned.
Determination of LDL cholesterol by beta-quantification is recognized as
the reference method but the procedure is so cumbersome relatively few
laboratories use this method. A recent
method using immunoseparation has become popular. However, this method still requires sample
pretreatment prior to cholesterol determination, making it unsuitable for full
automation of the procedure. The method
presented here offers direct determination of LDL cholesterol in a two part,
liquid stable reagent that is easily adapted to most automated chemistry
analyzers.
2.2 TEST PRINCIPLE
The LDL Cholesterol Reagent
is a two-part, liquid stable method for directly measuring LDL-C levels in
serum or plasma. The method depends on
the properties of a unique detergent, which eliminates the need for any off-line
pretreatment or centrifugation steps.
This detergent (Reagent 1) solubilizes only the non LDL lipoprotein
particles. The cholesterol released is
consumed by cholesterol esterase and cholesterol oxidase in a non-color forming
reaction. A second detergent (Reagent
2) solubilizes the remaining LDL particles and a chromogenic coupler allows for
color formation. The enzyme reaction
with LDL-C in the presence of the coupler produces color that is proportional
to the amount of LDL cholesterol present in the sample.
HDL, VLDL Solubilized HDL Consumed HDL, VLDL Chylomicrons
Chylomicrons VLDL, Chylomicrons (No color
development)
Detergent/Reagent 1 Detergent/Reagent 2
LDL
Non-solubilized
Solubilized
LDL
Cholesterol LDL Cholesterol
Solubilized LDL Cholesterol + H2O2
Cholesterol
Esterase
Cholesterol
Oxidase
H2O2 + DSBmT + 4-AA
Color Development (Measured
Biochromatical at 546 & 660nm)
Peroxidase
3.0 SPECIMEN COLLECTION AND STORAGE
3.1 SPECIMEN COLLECTION
Serum, EDTA-treated or
heparinized plasma are the recommended specimens. Patients are not required to fast prior to
blood collection.
Serum: Collect whole blood by venipuncture and allow to clot.
Centrifuge and remove the serum as soon as possible after collection (within 3
hours) (11.10).
Plasma: Specimens may be collected in EDTA or
heparin. Centrifuge and remove the plasma as soon as possible after collection
(within 3 hours) (11.10).
3.2 SPECIMEN STORAGE
If not analyzed promptly,
specimens may be stored at 2-8°C for up to 5 days. If specimens need to be stored for more that
5 days, they may be stored frozen at -80°C.
3.3 INTERFERENCES
All interference studies were
conducted according to the procedures recommended in NCCLS guideline No. EP7-P
for interference testing in clinical chemistry(11.12). Hemoglobin at levels up to 400 mg/dl and
Bilirubin at levels up to 20 mg/dl were found to exhibit negligible
interference (<5%) on this method.
Samples with levels of interfering substances higher than the upper
limits should be diluted with physiological saline before assaying. Multiply the result obtained from the manual
dilution by the appropriate dilution factor.
For a comprehensive review of drug interference on serum LDL cholesterol
levels see Young et al(11.13)
4.0 MATERIALS (1 X 30 ml, 1 X 10 ml)
(1 X 60 ml, 1 X 20 ml)
The reagents necessary for
the determination of LDL Cholesterol are included in the kit.
4.1 REAGENT COMPOSITION
Components Appearance Ingredients
Reagent 1 Liquid MES
Buffer (pH 6.3)
Detergent
1
Cholesterol
esterase
Cholesterol
oxidase
Peroxidase
4-aminoantipyrine
Ascorbic
acid oxidase
Preservative
Reagent 2 Liquid MES
Buffer (pH 6.3)
Detergent
2
N,N-bis(4-sulfhobutyl)-m-
Toluidine-disodium
(DSBmT)
Preservative
Cholesterol Oxidase from
Nocardia sp., Cholesterol Esterase from Pseudomonas sp., Peroxidase from
Horseradish, Ascorbic Acid Oxidase from Cucurbita sp.
4.2 WARNINGS AND
PRECAUTIONS
4.2.1 For In Vitro diagnostic use.
4.2.2 Do not pipette by mouth.
4.2.3 All specimens used in this test should be considered
potentially infectious. Universal precautions as they apply to your facility
should be used for handling and disposal of materials during and after testing.
4.2.4 Do not use the reagent after the expiration date printed on
the kit.
4.3 WORKING REAGENT
PREPARATION
Reagent 1: Reagent 1 is ready to use as packaged.
Reagent 2: Reagent 2 is ready to use as packaged..
4.4 REAGENT STORAGE AND STABILITY
All unopened reagents are
stable until the expiration date on the kit label when stored at 2-8°C.
4.5 ADDITIONAL
MATERIALS REQUIRED
4.5.1 LDL Cholesterol (Direct) Calibrator,.
4.5.2 Lipid Control,.
4.5.3 Automated clinical chemistry analyzer
capable of accommodating two-reagent assays.
5.0 PROCEDURE
Below is a general example of
the LDL test procedure for an automated analyzer. All analyzer applications should
be validated in accordance with NCEP and CLIA recommendations (11.10). For
assistance with applications on automated analyzers, please contact Biotron
Diagnostics technical service department at 1-800-595 8766.
37°C 37°C
Sample + Reagent 1 Reagent 2 Measurement
3µl 300µl 5 min 100µl 5 min of Absorbance 2
(660/546nm)
Measurement
of
Absorbance 1
(660/546nm) LDL-C Result
6.0 LIMITATIONS
6.0.1 Anticoagulants containing citrate should not be used.
6.0.2 Protect the reagents from direct
sunlight.
6.0.3 Samples with values greater than 700mg/dl must be diluted
with 1:1 with saline and reassayed.
Multiply the results by two.
7.0 CALIBRATION
The LDL Cholesterol
Calibrator is required for calibration.
The value of the LDL Calibrator was assigned by procedures traceable to
the National Reference System for Cholesterol (NRS/CHOL). Refer to LDL Cholesterol Calibrator package
insert for instructions. If control
results are found to be out of range the procedure should be recalibrated.
8.0 QUALITY CONTROL
Reliability of test results
should be routinely monitored with control materials that reasonably emulate
performance on patient specimens (11.10).
Quality control materials are intended for use only as monitors of
accuracy and precision. The recovery of
control values within the appropriate range should be the criteria used in
evaluation of future assay performance.
Controls should be run with every working shift in which LDL-C assays
are performed. It is recommended that
each laboratory establish their own
frequency of control determination.
Quality control requirements
should be performed in conformance with local, state, and/or Federal
regulations or accreditation requirements.
9.0 RESULTS
To convert from conventional
units to S.I. units, multiply the conventional units by 0.02586.
Example: mg/dl X
0.02586=mmol/L LDL-C
9.1 EXPECTED VALUES
The following NCEP
recommendations for patient classifications are suggested for the prevention
and management of coronary heart disease(11.8).
LDL
Cholesterol Classification
<130mg/dl
(3.36mmol/L) Desirable
130-159mg/dl
(3.36-4.11mmol/L) Borderline
High Risk
ł160mg/dl (4.14mmol/L) High
Risk
It is highly recommended that
each laboratory establish its own range of expected values.
10.0 SPECIFIC PERFORMANCE CHARACTERISTICS
10.1 ASSAY
RANGE: 0-700 mg/dl.
10.2 ACCURACY
Accuracy of the LDL
Cholesterol Reagent method was verified by comparison to the reference method (b-quantification followed by
cholesterol analysis),(11.10) another automatable LDL cholesterol method, and
the LDL immunoseparation method. Studies
comparing the LDL Cholesterol method to the reference method produced the
following results:
LDL Reference
Method Cholesterol Method
n 40 40
Mean LDL Cholesterol (mg/dl) 130.7 136.7
Range (mg/dl) 18-231 24-251
Standard Deviation (mg/dl) 43.8 45.9
Regression Analysis y=0.92x +
5.22mg/dl
Correlation Coefficient r=0.962
Studies comparing LDL
Cholesterol method to the other automatable LDL Cholesterol method produced the
following results:
LDL Automatable
Method Cholesterol Method
n 45 45
Mean LDL Cholesterol (mg/dl) 106.2 113.6
Range (mg/dl) 15-186 19-192
Standard Deviation (mg/dl) 33.9 36.0
Regression Analysis y=0.93x +
0.41mg/dl
Correlation Coefficient r=0.988
Studies comparing LDL
Cholesterol method to the LDL Cholesterol immunoseparation method produced the
following results:
LDL Immunoseparation
Method Cholesterol Method
n 31 31
Mean LDL Cholesterol (mg/dl) 117.9 120.4
Range (mg/dl) 50-219 39-231
Standard Deviation (mg/dl) 40.0 42.0
Regression Analysis y=0.90x +
9.61mg/dl
Correlation Coefficient r=0.944
10.3 PRECISION
Within-day precision for the
direct HDL Cholesterol Reagent was determined following a modification of NCCLS
document EP5-T2(11.17). Within-Day
precision studies produced the following results:
Sample LOW MID HIGH
n 20 20 20
Mean HDL Cholesterol (mg/dl) 37 122 187
Standard Deviation (mg/dl) 1.5 4.2 6.3
Coefficient of Variation (%) 4.1 3.4 3.4
Day- to-Day precision was
also determined following a modification of NCCLS document EP5-T2(11.17) Day-to-Day precision studies produced the following
results.
Sample LOW MID HIGH
n 20 20 20
Mean HDL Cholesterol (mg/dl) 38 135 222
Standard Deviation (mg/dl) 2.1 7.9 7.5
Coefficient of Variation (%) 5.4 5.9 3.4
10.4 SENSITIVITY
The analytical sensitivity
for LDL Cholesterol was determined to be 0.0013 absorbance units per 1mg/dl of
LDL cholesterol.
11.0 REFERENCES
11.1 Gotto A.M.,
Lipoprotein metabolism and the etiology of hyperlipidemia, Hospital practice,
23:Suppl. 1,4 (1988).
11.2 Crouse J.R.
et al., Studies of low density lipoprotein molecular weight in human beings
with coronary artery disease, J. Lipid Res., 25;566(1985).
11.3 Badimon
J.J., Badimon L., Fuester V., Regression of Atherosclerotic Lesions by
High-Density Lipoprotein Plasma Fraction in the Cholesterol-Fed Rabbit, Journal
of Clinical Investigation, 1990; 85:1234-41.
11.4 Castelli
W.P. et al., Cholesterol and other lipids in coronary heart disease,
Circulation, 55;767 (1977).
11.5 Barr D.P.,,
Russ E.M., Eder H.A., Protein-lipid relationships in human plasma, Am. J. Med.,
11;480 (1951).
11.6 Gordon T. et
al., High density lipoprotein as a protective factor against coronary heart
disease, Am. J. Med., 62;707 (1977).
11.7 Williams P.,
Robinson D., Baily A., High density lipoprotein and coronary risk factor,
Lancet, 1;72 (1979).
11.8 Kannel W.B.,
Castelli W.P., Gordon T., Cholesterol in the prediction of atherioslcerotic
disease; New perspectives based on the Framingham study, Am. Intern. Med.,
90;85 (1979).
11.9 National
Institutes on health publication No. 93-3095, September 1993.
11.10 Warnick G. Russell, Wood Peter D., National Cholesterol
Education Program Recommendations for Measurement of High-Density Lipoprotein
Cholesterol; Executive Summary, Clinical Chemistry, Vol. 41, No. 10, 1995.
11.11 Grundy S.M. et al., Summary of the Second Report of the
National Cholesterol Education Program (NCEP) Expert Panel on Detection,
Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment
Panel II), JAMA 1993, 269;23;3015-3023.
11.12 National Committee for Clinical Laboratory Standards,
National Evaluation Protocols for Interference Testing, Evaluation Protocol
Number 7, vol. 4, No. 8, June 1984.
11.13 Young, D.S., Effects of Drugs on Clinical Laboratory Tests,
3rd ed., AACC Press, Washington DC, 1990, 3-104 thru 3-106.
11.14 Tietz N.W.,, Clinical Guide to Laboratory Tests, W.B.
Saunders Co., Philadelphia, 1986, p. 256.
11.15 Carey RN , Garber CC. Evaluation of Methods. In Kaplan LA,
Pesce AJ, eds. Clinical Chemistry: theory, analysis and correlation. Third Edition.st.Louis: The CV Mosby Company.
11.16 Westgard JO, Carey RN. Wold S. Criteria
for judging precision and accuracy in method development and evaluation.
Cliical Chemistry 1974;20:825-833.
11.17 NCCLS document “Evaluation of Precision
Performance of Clinical Chemistry Devices” 2nd Ed. 1992.