N.S.

BIO-TEC CHOLESTEROL (CHOD-PAP)

Enzymatic Colorimetric Determination of Serum Cholesterol

REF. CHOL-MC – 0530 (5 x 30 ml)

INTENDED USE
NS Biotec cholesterol reagent is intended for the in vitro quantitative REAGENTS
determination of total cholesterol in serum and plasma on both automated Reagent Preparation & Stability
and manual systems. R1 Cholesterol standard 200 mg/dl

CLINICAL SIGNIFICANCE R2 Pipes buffer,

Cholesterol is a steroid with a secondary hydroxyl group in the C3 position, pH 6.9 90 mmol/l
Phenol 26 mmol/l
and found in blood, bile, and brain tissue. It serves as a precursor to bile
Cholesterol oxidase 500 U/l
acids, steroids and vitamin D. It is synthesized in many types of tissue, but Cholesterol esterase 500 U/l
particularly in the liver and intestinal wall. Approximately, 75% of cholesterol Peroxidase 1250 U/l
is newly synthesized and a 25% originates from dietary intake. 4-Aminoantipyrine 0.4 mmol/l
Measurements of serum cholesterol levels are important in the diagnosis
and classification of hyperlipoproteinemias. Elevated cholesterol levels may All reagents are ready for use and stable up to the expiry date given
0
on label when stored at 2–8 C.
occur with hypothyroidism, nephrotic syndrome, diabetes, and various liver
diseases. There is a correlation between elevated serum cholesterol levels SPECIMEN
and the incidence of coronary artery diseases. Normal cholesterol levels are • Serum, or plasma.
affected by stress, diet, age, gender, hormonal balance, and pregnancy. • The only acceptable anticoagulants are heparin and EDTA.
Depressed levels are associated with hyperthyroidism and severe liver
Specimen Preparation & Stability
diseases.
No special preparation of the patient is necessary, however it is
recommended that prior to collection, patients should be following
ASSAY PRINCIPLE their usual diet and be in their usual state of health. Patients who are
Cholesterol analysis was first reported by Liebermann in 1885 followed by acutely ill, losing weight, pregnant or have had a myocardial infarction
Burchard in 1889. In Liebermann-Burchard reaction, cholesterol forms a in the previous 3 months should be rescheduled.
blue-gree dye from polymeric unsaturated carbohydrates in an acetic
acid/acetic anhydride/concentrated sulfuric acid medium. The Abell and Blood should be collected by venipuncture, after the patient has been
Kendall method is specific for cholesterol but is technically complex and in a seated position for at least 5 minutes. Tourniquet usage should
requires corrosive reagents. In 1974, Allain et al and Roeschlau et al were be kept to a minimum and the specimen should be allowed to clot for
able to combine cholesterol esterase and cholesterol oxidase into a single 30 minutes at room temperature.
enzymatic reagent for the determination of total cholesterol. NS Biotec The best specimen is unhemolysed serum, and should be analyzed
cholesterol reagent combines the use of these enzymes with the 0
on the day of collection. When stored at 4 C, specimens are stable for
11
peroxidase/phenol/4-aminoantipyrine system of Trinder for the 0
3-4 days; specimens are stable at –20 C for several months.
measurement of total cholesterol in human serum.
The series of reactions involved in the assay system are as follows: PROCEDURE

1. Cholesterol esters are enzymatically hydrolyzed by cholesterol • Manual Procedure

esterase (CE) to cholesterol and free fatty acids. Wavelength 500 - 550 nm
2. Free cholesterol, including that originally present, is then oxidized Cuvette 1 cm light path
by cholesterol oxidase (CHOD) to cholest-4-en-3-one and H2O2. Temperature
0
20-25 or 37 C
3. In presence of peroxidase (POD), the formed hydrogen peroxide Zero adjustment against reagent blank
formed effects the oxidative coupling of phenol and 4-
aminoantipyrine (4-AAP) to form a red-colored quinoneimine dye. Specimen Serum or plasma

CHOD
Cholesterol esters+H2O Cholesterol + fatty acids
CE Blank Standard Specimen
Cholesterol +O2 Cholest-4-ene-3-one+H2O2
POD
2 H2O2+ 4-AAP + Phenol Quinoneimine dye + 4 H2O R2 1.0 ml 1.0 ml 1.0 ml
Standard …… 10 µl ……
The intensity of the color produced is directly proportional to cholesterol Specimen …… …… 10 µl
concentration. It is determined by measuring the increase in absorbance at
500 – 550 nm.
0 0
EXPECTED VALUES Mix, incubate for 5 minutes at 37 C or 10 minutes at 20-25 C. Measure
the absorbance of specimen (Aspecimen) and standard (Astandard) against
reagent blank.
Risk classification Total Cholesterol
The color is stable for 60 minutes.
Desirable <200 mg/dl (5.2 mmol/l)
Borderline high 200 – 239 mg/dl • Automated Procedure
(5.2 – 6.2 mmol/l) User defined parameters for different auto analyzers are available
High >240 mg/dl (6.2 mmol/l) upon request
CALCULATION
Each laboratory should investigate the transferability of the expected Calculate the cholesterol concentration by using the following
values to its own patient population and if necessary determine its own formulae:
reference range. For diagnostic purposes, the cholesterol results
Cholesterol Concentration=
should always be assessed in conjunction with the patient’s medical
history, clinical examination, and other findings. Absorbance of Specimen X Standard value
Absorbance of Standard

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 Unit conversion BIBLIOGRAPHY
mg/dl x 0.0259 = mmol/l
1. Searcy, RL (1969): Diagnostic Biochemistry. McGraw-Hill, New York, NY.
LINEARITY
2. Ellefson, RD and Garaway, WT (1976): Fundamentals of clinical
When run as recommended, the assay is linear up to 800 mg/dl (20.7 chemistry. Ed Tietz NW, p 506.
mmol/l).
3. Lieberman, C. (1885): Ber. 18: 1803.
If result exceeds 800 mg/dl (20.7 mmol/l), specimen should be diluted
4. Burchard, H (1890): Chem. Zentr. 61: 25.
with 0.9% NaCl solution and reassayed. Multiply the result by the
5. Flegg, HM (1973): Ann. Clin. Biochem. 10: 79.
dilution factor.
6. Richmond, W (1972): Scand. J. Cal. Invest. 29 (Suppl): 126.
SENSITIVITY
7. Hernandez, HH and Chaikoff, IL (1957): J. Biol. Chem. 228: 447.
The sensitivity is defined as the change of analytical response per unit
change in analyte concentration at a pathlength of 1 cm. 8. Hyun, J, et al., (1969): J. Biol. Chem. 244: 1937.
When run as recommended the sensitivity of this assay is 3.0 mg/dl 9. Allain, CC, et al., (1974): Clin. Chem. 20: 470.
(0.08 mmol/l). 10. Roeschlau, P, Bernt, E and Gruber, F (1974): Clin. Chem. Clin.
Biochem. 12: 226.
QUALITY CONTROL
11. Trinder, P (1969): Ann. Clin. Biochem. 6: 24.
It is recommended that controls (normal and abnormal) be included in:
12. Henry, RJ (1974): Clinical Chemistry: Principles and Techniques.
• Each set of assays, or Harper & Row, Hagerstown, MD.
• At least once a shift, or
13. Young, DS (1990): Effects of Drugs on Clinical Laboratory Tests. Third
• When a new bottle of reagent is used, or Edition. 1990: 3: 6-12.
• After preventive maintenance is performed or a clinical component
14. Young, DS, et al., (1975): Clin. Chem. 21: 5.
is replaced.
Commercially available control material with established cholesterol
values may be routinely used for quality control.
Failure to obtain the proper range of values in the assay of control material
may indicate:
• Reagent deterioration,
• Instrument malfunction, or
• Procedure errors.
The following corrective actions are recommended in such situations:
• Repeat the same controls.
• If repeated control results are outside the limits, prepare fresh
control serum and repeat the test.
• If results on fresh control material still remain outside the limits,
then repeat the test with fresh reagent.
• If results are still out of control, contact NS Biotec Technical
Services.
INTERFERING SUBSTANCES
• Anticoagulants
The only acceptable anticoagulants are heparin and EDTA.
• Bilirubin:
Bilirubin levels higher than 7.5 mg/dl decrease the apparent total
cholesterol concentration significantly.
• Drugs:
Methyldopa causes artificially low total cholesterol values at the
tested drug level. For a more comprehensive review of drugs
13
affecting cholesterol assays refer to the publication by Young .
• Haemoglobin:
No interference from haemoglobin up to a level of 500 mg/dl.
• Lipemia:
No significant interference.
• Others: N.S BIOTEC
Ascorbic acid levels higher than 7.5 mg/dl decrease the apparent
MEDICAL EQUIPMENTS
total cholesterol concentration significantly.
Other 3-beta-hydroxysteroids cause positive interference but are
7 Sad Zaghloul sq. Mehatet Elraml, Wellkang Ltd
14
not normally present in significant quantities in human serum . Alexandria – Egypt suite B , 29 Harley
WARNING & PRECAUTIONS Tele: 002 03 486 5140 street LONDON ,
Fax: 002 03 485 7597 W 1 G 9 QR , U.K.
• NS Biotec cholesterol reagent is for in vitro diagnostic use only. Website : www.nsbiotec.com www.CE-marking .EU
Normal precautions exercised in handling laboratory reagents should E- mail : [email protected]
be followed.
• Warm up working solution to the corresponding temperature before
use.
• The reagent and sample volumes may be altered proportionally to
accommodate different spectrophotometer requirements.
• Valid results depend on an accurately calibrated instrument,
timing, and temperature control.
• The reagent blank will not exceed an absorbance of 0.06 but
don’t use the reagent if it is turbid or if the absorbance is
greater than 0.2 at 500 nm.

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