1.0 INTENDED USE
This reagent
is intended for the quantitative determination of high-density lipoprotein
cholesterol (HDL-C) in human serum or plasma.
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, while the
inner core contains mostly esterified cholesterol and triglyceride. These
particles serve to solubilize and transport cholesterol and triglyceride in the
bloodstream.
The relative
proportions of protein and lipid determine the density of these lipoproteins
and provide a basis on which to begin their classification (11.1). These
classes are: chylomicron, very-low-density lipoprotein (VLDL), low-density
lipoprotein (LDL) and high-density lipoprotein (HDL). Numerous clinical studies
have shown that the different lipoprotein classes have very distinct and varied
effects on coronary heart disease risk (11.2).
The
principle role of HDL in lipid metabolism is the uptake and transport of
cholesterol from peripheral tissue to the liver through a process known as
reverse cholesterol transport (a proposed cardioprotective mechanism) (11.3).
Low HDL-C levels are associated with an increased risk of coronary heart
disease and coronary artery disease (11.4-11.9). Hence, the determination of
serum HDL-C is a useful tool in identifying high-risk patients. The Adult
Treatment Panel of the National Cholesterol Education Program (NCEP) recommends
that all adults 20 years of age and over should have their total cholesterol
and HDL cholesterol levels measured at least every 5 years to screen for
coronary heart disease risk (11.9).
The CDC
reference method for the quantitation of HDL-C combines ultracentrifugation and
chemical precipitation to separate HDL from other lipoproteins, followed by
cholesterol measurement using the Abell-Kendall assay (11.10). This method is
too time consuming and labor intensive for use in routine analysis (11.11).
Therefore, most laboratories utilized one of several methods for selective
precipitation and removal of LDL and VLDL, followed by the enzymatic
measurement of HDL-C in the supernatant fraction (11.10). Since these methods
require off-line pretreatment and separation steps the assay procedures cannot
be fully automated. As a result, routine determination of HDL-C has suffered
from long handling times and poor reproducibility.
2.2 TEST
PRINCIPLE
This HDL
Cholesterol assay is a homogeneous method for directly measuring serum HDL-C
levels without the need for any off-line pretreatment or centrifugation steps.
The method
is in a two-reagent format (illustrated below). The first reagent contains a
mixture of polymers and polyanions that bind to the surface of LD, VLDL, and
chylomicrons. These complexed lipoproteins are stabilized even in the presence
of detergent, which is added as part of the second reagent, together with the
remaining components of a cholesterol reagent. HDL particles on the other hand
are not stabilized by the polymers and polyanions and become solubilized by the
detergent. Consequently, only the HDL Cholesterol is subject to cholesterol
measurement.
3.0 SPECIMEN COLLECTION AND STORAGE
3.1 SPECIMEN
COLLECTION
Serum,
EDTA-treated or heparinized plasma are the recommended specimens.
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).
Samples
containing triglyceride levels of > 2,871 mg/dl should be diluted with one
part sample to one part physiological saline before assaying. Multiply the
result by two.
3.2 SPECIMEN
STORAGE
If not
analyzed promptly, specimens may be stored at 2-8°C for up to 1 week. If
specimens need to be stored for more than 1 week, they may be preserved at less
than -20°C for up to one month. For storage periods of 1 month to 2 years,
samples should be preserved at -70°C (11.10).
4.0 MATERIALS (1 x
30 ml, 1 x 10 ml)
(2 x 60 ml, 2 x 20 ml)
The reagents
necessary for the determination of HDL Cholesterol are included in the kit.
4.1 COMPOSITION
OF REAGENTS
Reagent 1: a-cyclodextrin 0.5 mM, dextran sulfate 0.5 g/l, magnesium
chloride 2.0 mM, HSDA 0.3 g/l, buffer, pH 7.0 ± 0.1,
preservative. aPolyanion Neutralizes ionic charges on the surface of
LDL thereby strengthening the binding with the polymer.
Reagent 2:
POD > 15,000 U/L, PEG-CO > 5,000 U/L, PEG-CE > 800 U/L,
4-aminoantipyrene 0.5 g/l, buffer, pH 7.0 ± 0.1,
surfactant, preservative.
HSDA =
sodium N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline.
PEG-CO=Cholesterol
Oxidase from Nocardia sp.
PEG-CE=Cholesterol
Esterase from Pseudomonas
POD=Peroxidase
from Horseradish
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.
Reagent 2: Reagent 2 is ready to use.
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 HDL Cholesterol (Direct) Calibrator.
4.5.2 Tri-level
Lipid Control,
4.5.3 4.5.3 Automated clinical chemistry analyzer
capable of accommodating two-reagent assays.
5.0 PROCEDURE
Below is a
general example of the HDL 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
4ml 300ml 5 min 100ml 5 min (absorbance
difference
between
700nm & 600nm)
HDL-C Result
6.0 CALIBRATION
The value of
the HDL Calibrator was assigned by procedures traceable to the National
Reference System for Cholesterol (NRS/CHOL).
Calibration materials have concentrations around the medical decision
level. Refer to HDL Cholesterol
Calibrator kit package insert for instructions.
7.0 INTERPRETATION OF RESULTS
7.1 EXPECTED
VALUES
The range of
expected values is (11.14):
Males: 30-70mg/dl
Females: 30-85mg/dl
Each
laboratory must establish its own range of expected values.
According to
the NCEP, HDL values greater than or equal to 35mg/dl are considered desirable,
and values greater than or equal to 60mg/dl are considered to offer some
protection against coronary heart disease.
Values below 35mg/dl are considered to be a significant independent risk
factor for coronary heart disease (11.9).
7.2 SI UNITS
To convert
from conventional units to S.I. units, multiply the conventional units by
0.02586.
mg/dl X 0.02586=mmol/L HDL-Cholesterol
7.3 LIMITATIONS
7.3.1 Anticoagulants containing citrate should
not be used.
7.3.2 Protect the reagents from direct sunlight.
7.3.3 Store the
reagents as per instructions.
7.4.3 Samples with values greater than 150 mg/dl
must be diluted 1:1 with saline and reassyed. Multiply the result by 2.
8.0 INTERFERING SUBSTANCES
All
interference studies were conducted according to NCCLS guideline No. EP7-P for
interference testing in clinical chemistry (11.12).
Hemoglobin
levels up to 100 mg/dl and bilirubin levels up to 20 mg/dl were found to
exhibit negligible interference (<5%); interference was observed at 400
mg/dl.
Samples with
levels of interfering substances higher than the upper limits should be diluted
with physiological saline before assaying.
Refer to the
work of Young for a review of drug effects on serum HDL Cholesterol levels
(11.13).
9.0 QUALITY CONTROL
Reliability
of test results should be routinely monitored with control sera or
quality-control materials that reasonable emulate performance on patient
specimens (11.10). Quality control
materials are intended for use only as monitors of accuracy and precision. The National Cholesterol Education Program
(NCEP) Lipid Standardization Panel (LSP) recommends two levels of controls, one
in the normal range (35-65mg/dl) and one near the concentrations for decision
making (<35mg/dl). An acceptable
range of HDL Cholesterol values should be established for the controls by
repeat analysis. The recovery of control
values within the appropriate range should be the criteria used in evaluation
of future assay performance.
Quality
control requirements should be performed in conformance with local, state,
and/or Federal regulations or accreditation requirements.
10.0 SPECIFIC
PERFORMANCE CHARACTERISTICS
10.1 ACCURACY
Accuracy of
the direct HDL Cholesterol Reagent method was verified by comparison to the
designated comparative method (ultracentrifugation, chemical precipitation and
Abell-Kendall cholesterol analysis) (11.10) and the Biotron lyophilized direct
HDL method. Studies comparing the HDL
Cholesterol method to the designated comparative method produced the following
results:
Direct Designated
HDL Comparative
Method Cholesterol Method
n 30 30
Mean (mg/dl) 55 55
Range
(mg/dl) 26-94 26-94
Standard
Deviation (mg/dl) 18 19
Regression
Analysis y=0.93x -
3.85
Correlation
Coefficient r=0.989
Lyophilized
Direct
HDL Direct HDL
Method Cholesterol Method
n 95 95
Mean (mg/dl) 59 59
Range (mg/dl) 27-109 24-118
Standard
Deviation (mg/dl) 20 21
Regression
Analysis y=0.91x +
6.08
Correlation
Coefficient r=0.979
10.2 PRECISION
Within-day
precision for the direct HDL Cholesterol Reagent was determined following a
modification of NCCLS document EP5-T2 (11.15.)
Within-day precision studies produced the following results:
Low Medium High
n 20 20 20
Mean (mg/dl) 38 68 85
Standard
Deviation (mg/dl) 0.9 1.0 1.2
Coefficient
of Variation (%) 2.4 1.5 1.4
Day-to-day
precision was determined following a modification of NCCLS document EP5-T2
(11.15.) Day-to-day precision studies
produced the following results:
Low Medium High
n 20 20 20
Mean (mg/dl) 37 66 84
Standard
Deviation (mg/dl) 0.8 1.5 1.6
Coefficient
of Variation (%) 2.2 2.3 1.9
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 NCCLS document “Evaluation of Precision
Performance of Clinical Chemistry Devices” 2nd Ed. 1992.