HDL PRECIPITATING REAGENT PRINCIPLE OF THE METHOD TEST PERFORMANCE

Polyethylene glycol, average MW 6000, in acqueous solu-
CD 0400 CH 4 x 100 ml
SUMMARY OF TEST
Although the metabolism of chylomicrons, VLDL, IDL, and
LDL is reasonably well understood, our knowledge about
HDL is relatively new and still growing. HDL consists of a
number of polydisperse and heterogeneous particles that
vary with respect to size and content of lipid and apolipopro-
tein. HDL can be separated not only by ultracentrifugation
and electrophoresis but also by polyanionic precipitation.
About 50% of HDL mass is protein, 30% is phospholipid,
and 20% is cholesterol. The major apolipoproteins found in
HDL are A-I and A-Il and constitute about 90% of total HDL
protein. The ratio of apo A-I to apo A-IT is approximately
3: 1 by weight.
Both the liver and the intestine are involved in the production
of HDL, although the exact roles and relative importance
of each are not fully understood. The half-life of HDL in
plasma in normal subjects is approximately 4 days. Little
is known about the sites of HDL catabolism; however, the
liver and kidneys are probably involved.
HDL appears to play an important part in cholesterol efflux
from tissues, thereby reducing the amount of cholesterol
stored there; this is apparently mediated by a particular
lipoprotein particle containing only apo A-I, known as
LpA-I. HDL also has a role in returning cholesterol from the
periphery to the liver for removal as bile acids, a process
known as reverse cholesterol transport. Evidence tends to
support the suggestion that HDL serves as a scavenger
of lipid and apolipoprotein during the normal catabolism
of chylomicrons and VLDL. HDL acquires free cholesterol
released from these molecules, and plasma LCAT converts
this free cholesterol to its esters (with fatty acids derived
from lecithin). These esters are later transferred back to
VLDL and IDL by way of apo D or a protein known as
lipid transfer protein. HDL is also known to function as
a plasma reservoir for apo C-Il. The relationship of HDL
and VLDL with chylomicrons is exemplified by the fact
that defects in triglyceride-rich lipoprotein catabolism are
commonly associated with marked reduction in HDL levels.
Epidemiological studies have suggested that HDL protects
against cardiovascular disease, and a significant amount of
research has been devoted to HDL in order to demonstrate
its role in reverse cholesterol transport.
The importance of HDL-C as an independent risk factor
for developing CAD is now recognized by the scientific
community. An outcome from an NIH consensus conference
on the relation of HDL-C to CAD recommended that
HDL levels, in addition to total cholesterol, be routinely
measured when evaluating patients for CAD risk. This
recommendation clearly reflects the new epidemiological
data that suggest that low HDL level is a common and
strong risk factor for CAD in both men and women, that it
can and should be modified, and that it still can be a factor
even when total cholesterol levels are desirable. Because it
currently is extremely difficult and impractical to quantitate
HDL directly, most methods depend on the measurement
of the plasma content of HDL-C. The majority of techniques
are based on selective precipitation of VLDL and LDL
with various polyanions, followed by measurement of the
cholesterol concentration in the supernatant containing
the HDL.
Numerous studies have compared the different methods
for measuring HDL-C. Isolation procedures involving
preparative ultracentrifugation are considered reference
methods and are generally used only in specialized
research centers. Various precipitation techniques are
now recommended. Techniques for preparative isolation
of the lipoprotein classes by precipitation have now been
adopted in routine clinical chemistry for the separation of
HDL in small volumes of plasma. The lipoprotein classes
can be selectively precipitated by appropriately chosen
polyanions. For clinical purposes, a reagent-precipitating
system must form an insoluble complex with all the plasma
lipoproteins except HDL so that HDL remaining in the
supernatant after centrifugation can be quantitated by its
cholesterol content.
The present reagent uses a solution of polyethyleneglycole
with an average MW of 6000 (PEG 6000). A determined
concentration of this polyanion could get precipitation of
various macromolecules, including lipoproteins. After pre-
cipitation and centrifugation, the HDL fraction of cholesterol
is measured in the supernatant.
Sample is prepeared through precipitation procedures as
tion, is used to precipitate lipoproteins VLDL and LDL. After
centrifugation, the clear supernatant containing HDL frac-
tion is suitable for enzymatic determination of cholesterol.
KIT COMPONENTS
For in vitro diagnostic use only.
The components of the kit are stable until expiration date
on the label.
Keep away from direct light sources.
Reagent A: 4 x 100 ml (liquid) blue cap
Composition: polyethylenglycol 16 %, non reactive additi-
ves and stabilizers.
Store all components at 2-8°C.
MATERIALS REQUIRED BUT NOT SUPPLIED
Current laboratory instrumentation. Spectrophotometer
UV/VIS with thermostatic cuvette holder. Automatic micro-
pipettes. Glass or high quality polystyrene cuvettes. Saline
solution.
The kit CT F400 CH or CT 150F CH CHOLESTEROL FL
and your STANDARD are needed to perform the colorime-
tric assay run.
REAGENT PREPARATION
Use reagent ready to use.
Stability: up to expiration date on labels at 2-8°C.
Stability since first opening of vials: preferably within 60
days at 2-8°C.
PRECAUTIONS
Reagent may contain some non-reactive and preservative
components. It is suggested to handle carefully it, avoiding
contact with skin and swallow.
Perform the test according to the general “Good Labora-
tory Practice” (GLP) guidelines.
SPECIMEN
Serum, plasma EDTA.
Sample is stable 3 days at 2-8°C and 1 month at -20°C.
TEST PROCEDURE - PRECIPITATION STEP
Pipet in centrifuge tubes:
sample 500 µl + reagent 500 µl
mix by inversion, incubate 5 minutes, centrifuge at 3000
g/min for 10 minutes.
Separate supernatant and use it as sample into following
procedure.
TEST PROCEDURE - QUANTITATIVE STEP
Wavelenght: 510 nm (allowed 480 ÷ 520 nm)
Lightpath: 1 cm
Temperature: 37°C
dispense: blank standard sample
reagent 1 ml 1 ml 1 ml
water 25 µl - -
standard - 25 µl -
sample - - 25 µl
Mix, incubate at 37°C for 5 minutes.
Read absorbances of standard (As) and samples (Ax)
against reagent blank.
RESULTS CALCULATION
serum/plasma sample:
HDL cholesterol mg/dl = Ax/As x stand. value x 2
(standard value + dilution factor)
EXPECTED VALUES
high risk average low risk
Men: < 40 mg/dl 40-50 mg/dl > 50 mg/dl
Women: < 45 mg/dl 45-60 mg/dl > 60 mg/dl
Each laboratory should establish appropriate reference
intervals related to its population.
QUALITY CONTROL AND CALIBRATION
It is suggested to perform an internal quality control. For
this purpose a suitable human based control sera has to
be used.
indicated above, supernatant is analyzed by reagent CT
F400 CH - CT 150F CH
Linearity
the method is linear up to 700 mg/dl.
If the limit value is exceeded, it is suggested to dilute
sample 1+9 with saline and to repeat the test, multiplying
the result by 10.
Sensitivity/limit of detection (LOD)
the limit of detection is 1 mg/dl.
Interferences
no interference was observed by the presence of:
hemoglobin ≤ 500 mg/dl
bilirubin ≤ 15 mg/dl
lipids ≤ 850 mg/dl
Precision
intra-assay (n=10) mean (mg/dl) SD (mg/dl) CV%
sample 1 101.50 1.84 1.80
sample 2 176.20 2.74 1.60
inter-assay (n=20) mean (mg/dl) SD (mg/dl) CV%
sample 1 100.99 2.11 2.10
sample 2 176.51 2.23 1.30
Methods comparison
a comparison between Chema and a commercially availa-
ble product gave the following results:
Cholesterol FL Chema = x
HDL-direct competitor = y
n = 75
y = 0.980x + 0.608 mg/dl r2 = 0.957
WASTE DISPOSAL
This product is made to be used in professional laborato-
ries. Please consult local regulations for a correct waste
disposal.
S56: dispose of this material and its container at hazar-
dous or special waste collection point.
S57: use appropriate container to avoid environmental
contamination.
S61: avoid release in environment. Refer to special instruc-
tions/safety data sheets.
REFERENCES
Trinder P., - J. Clin. Path. 22, 158 (1969);
Allain C.C., Poon L.S., Chan C.S., Richmond W., Fu P.C.,
- Clin. Chem. 20,470 (1974).
National Cholesterol Education Program (NCEP) recom-
mended values for cholesterol
Tietz Textbook of Clinical Chemistry, Second Edition,
Burtis-Ashwood (1994).
MANUFACTURER
Chema Diagnostica
Via Campania 2/4
60030 Monsano (AN) - ITALY - EU
phone +39 0731 605064
fax +39 0731 605672
e-mail: mail@chema.com
website: http://www.chema.com
SYMBOLS

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IUS-7.5 UK rev. 23/05/2011