Cholesterol (Liquid)

Reagent Set

Intended Use Specimen Collection and Storage

For the quantitative determination of Total Cholesterol in serum. Nonhemolyzed serum is recommended. Cholesterol in serum is reported stable
for seven days at room temperature (18-25C) and six months when frozen and
Method History properly protected against evaporation.8,9
A Cholesterol method developed in the late 1800s by Lieberman1 and
Burchard2 is still in use today despite its corrosive nature and its Interferences
susceptibility to many interfering substances. A number of drugs and substances affect concentrations of cholesterol. See
Work on an enzymatic procedure was begun by Flegg3 and Richmond4 in the Young, et al.10
early 70s. Allain5 and Roeschlau6 began using cholesterol esterase and
oxidase, in a single reagent to determine total cholesterol in serum. Materials Provided
Trinders7 color system of peroxidase/phenol/4-aminoantipyrine has been Cholesterol Reagent.
used successfully for some time now. With appropriate calibrator value
assignment, this method has been shown to provide excellent accuracy in Materials Required but not Provided
relation to the reference methodology. 1. Accurate pipetting devices.
2. Timer.
Principle 3. Test tubes/rack
C. Esterase 4. Spectrophotometer with ability to read at 500 nm.
Cholesterol Esters -------------------------- Cholesterol + Fatty Acids 5. Heating Block (37C).

C. Oxidase Procedure (Automated)

Cholesterol + O2 ------------------------- Cholesterol-3-one + H2O2 Refer to specific instrument application instructions.

Peroxidase Procedure (Manual)

2H2O2 + 4-AAP + Phenol ------------------ Quinoneimine + 4 H2O
1. Label test tubes: Blank, Standard, Control, Patient, etc.
(red dye)
2. Pipette 1.0 ml of reagent into each tube and pre-warm at 37C for at least
five minutes.
The intensity of the red color produced is directly proportional to the total
3. Add 0.01 ml (10ul) of sample to respective tubes. Mix and return to 37C.
cholesterol in the sample when read at 500nm.
4. Incubate all tubes at 37C for five minutes.
5. Zero spectrophotometer with blank at 500nm.
Reagents 6. Read and record absorbances of all test tubes.
4-Aminoantipyrine 0.25mM, Cholesterol Esterase >150u/L, Cholesterol
Oxidase >150u/L, Peroxidase >1500u/L, Phenol >15mM, Phosphate Buffer, Procedure Notes
pH 6.8, non-reactive stabilizers and preservatives.
1. If the spectrophotometer being used requires a final volume greater than
1.0ml for accurate reading, use 0.025ml (25ul) of sample to 3.0ml of reagent.
Reagent Preparation Perform the test as described above.
The reagent is ready to use. 2. Grossly lipemic serums require a sample blank. Add 0.01ml (10ul) of
sample to 1.0ml saline, mix and read the absorbance against water.
Reagent Storage Subtract this value from the patient absorbance to obtain the corrected
1. Store reagent at 2-8C. reading.
2. The reagent is stable until the expiration date when stored at 2-8C.
Limitations
Reagent Deterioration Samples with values exceeding 500 mg/dl should be diluted 1:1 with saline and re-
Do not use if: run. The final answer should be multiplied by two.
1. The reagent is turbid.
2. The reagent does not meet stated performance parameters. Calibration
Aqueous standards can be used to calibrate the procedure or an appropriate
Precautions serum calibrator. The procedure should be calibrated according to the instrument
1. This reagent is for in vitro diagnostic use only. manufacturers instructions. If control results are found to be out of range, the
2. Not to be used internally in humans or animals. Normal precautions for procedure should be re-calibrated.
handling laboratory reagents should be followed.
3. Additional safety information concerning storage and handling of this Calculation
product is in the Material Safety Data Sheet for this product. Abs. = Absorbance

Abs. (Patient) x Concentration of Std. = Cholesterol (mg/dl)

Abs. (Standard) (mg/dl)
 Cholesterol (Liquid)
Reagent Set

Example: Abs. (Patient) = 0.40, Abs. (Standard) = 0.32, Concentration of

Standard = 200 mg/dl

0.40 x 200 = 250 mg/dl

0.32

Quality Control
Serum controls with known normal and elevated values should be run
routinely to monitor the validity of the reaction. These controls should be run
at least with every working shift in which Cholesterol 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.

Expected Values11
Recommended Range:
Desirable Cholesterol: <200mg/dl
Borderline-High Cholesterol: 200-239mg/dl
High Cholesterol: >240mg/dl

Performance
1. Linearity: 500 mg/dl
2. Comparison: A comparison between this procedure and a similar dry
powder reagent produced a regression equation of y = 1.02x + 0.8 with
a correlation coefficient of 0.999.
3. Precision:

Within Run Run to Run

Mean S.D. C.V.% Mean S.D. C.V.%
157 1.1 0.7 155 1.3 0.8
224 1.7 0.8 227 2.4 1.1 Manufactured by Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188
4. Specificity: Cholesterol oxidase is not totally specific for cholesterol.
Other analogs of cholesterol (dihydrocholesterol, 7-dehydrocholesterol, European Authorized Representative:
20-hydroxycholesterol, etc.) are also oxidized. These analogs do not Obelis s.a.
normally occur in any appreciable amounts in serum. Boulevard Gnral Wahis 53
1030 Brussels, BELGIUM
References Tel: (32)2.732.59.54 Fax: (32)2.732.60.03 email: mail@obelis.net
1. Lieberman, C., Ber. 18:1803 (1885).
2. Burchard, H., Chem. Fentr. 61:25 (1890).
3. Flegg, H.M., Ann. Clin. Biochem. 10:79 (1973).
4. Richmond, W., Scand. J. Clin. Lab. Invest. 29:Suppl. 26, abstr. 3:25
(1972).
5. Allain, C.C., et al, Clin. Chem. 20:470 (1974).
6. Roeschlau, P., et al, Z. Klin. Chem. Klin. Biochem 12:226 (1974). C7510 Manufactured by Pointe Scientific, Inc.
7. Trinder, P., Ann. Clin. Biochem. 6:24 (1969). 5449 Research Drive Canton, MI 48188
8. Perlstein, M.T., et al, J. Microchem. 22:403 (1977).
9. Witte, D.L., et al, Clin. Chem. 20:1282 (1974).
10. Young, D.S. et al, Clin. Chem. 21:1D (1975).
11. National Institute of Health Publication No. 88-2926 Detection,
Evaluation, and Treatment of High Cholesterol in Adults, November
(1987).
Rev. 5/17 P803-C7510-01

Use by (YYYY-MM-DD) Lot and batch code Catalog number Manufacturer In vitro diagnostic medical device

Temperature limitation Consult instructions for use CE mark Authorized representative in the European Community