TOTAL CHOLESTEROL (LIQUID) PROCEDURE

Intended for the Quantitative Determination of Serum Total Cholesterol

SUMMARY AND EXPLANATION

Total serum cholesterol has proved useful in the diagnosis of hyperlipoproteinemia, atherosclerosis, hepatic and thyroid diseases (1). Total and HDL cholesterols, in conjunction with a triglyceride determination, provide valuable information for the prediction of the coronary heart disease (2), and for lipoprotein phenotyping according to the Fredrickson classification (3).

This TOTAL CHOLESTEROL (LIQUID) PROCEDURE is based on a modified enzymatic method first described by Allain et al (4). P-Hydroxybenzene sulfonate replaces phenol in the formulation providing a safer, less corrosive reagent.

PRINCIPLE

The cholesterol assay involves sequential enzymatic reactions. Cholesterol esterase hydrolyzes cholesterol esters to cholesterol and free fatty acids. Cholesterol oxidase then oxidizes cholesterol to form cholest-4-en-3-one and hydrogen peroxide. Peroxidase catalyzes the hydrogen peroxide oxidation of 4-aminophenazone with subsequent coupling to p-hydroxybenzensulfonate. The end product is a quinoneimine dye which absorbs at 520 nm. The color intensity at 520 nm is directly proportional to serum cholesterol concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2520 provides:

CHOLESTEROL LIQUID REAGENT- (Cat. No. 2521)

REACTIVE INGREDIENTS:

20 mM p-Hydroxybenzenesulfonate, >>400 U/L Cholesterol Oxidase, >>400 U/L Cholesterol Esterase, >>5000 U/L Peroxidase, 10 MM 4-aminophenazone in a buffered solution with a preservative.

PRECAUTIONS:

Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if kept refrigerated and sealed tightly.

DETERIORATION:

The reagent should be a clear, dark yellow solution. Turbidity indicates contamination and the reagent should not e used. Developing a light pink color is normal, discard the reagent blank absorbance when measured against D-H20 >> 0.25 at 520 nm. Failure to achieve assayed values of freshly prepared control sera would also indicate deterioration and the reagent should be discarded.

CHOLESTEROL CALIBRATOR - (Cat. No. 2502)

REACTIVE INGREDIENTS:

200 mg/dL cholesterol in an aqueous solution that has been calibrated against an NIST traceable cholesterol standard.  

PRECAUTIONS:

Do not ingest. Causes irritation, avoid contact with skin, eyes and clothing. In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if kept tightly sealed to avoid evaporation.

DETERIORATION:

The calibrator should be a clear, colorless solution. Turbidity would indicate contamination.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 520 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Unhemolyzed serum collected after a 12 hour fast is recommended.

2. Plasma may be used if blood is collected in EDTA or heparin.

3. A non-fasting specimen may be used if the sample is not turbid.

4. Turbid / Lipemic samples require a Serum Blank.

SAMPLE STORAGE:

Serum cholesterol appears stable for 5 days at 2 - 8º C.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Bilirubin above the normal range will depress the cholesterol value approximately 2 mg/dL for each 1 mg/dL billirubin. Gentisic acid significantly affects the enzymatic cholesterol assay. Oxalate, flouride and citrate interfer with the procedure.

Young et al (5) have reviewed drug effects on serum cholesterol levels.

PROCEDURE

MATERIALS PROVIDED:

CHOLESTEROL LIQUID REAGENT (Cat. No. 2521) and CHOLESTEROL CALIBRATOR (Cat. No. 2502).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 1.0 mL pipettor or dispensor

2. 0.01 mL micropipettor

3. Incubator capable of maintaing 37º C

4. Timer, test tubes and rack

REACTION CONDITIONS:

Wavelength 520 nm

Filter Selection 500 - 550 nm

Reaction Type Endpoint

Reaction Time 10 minutes at 37º C

and Temperature 20 minutes at 30º C

25 minutes at 25º C

Sample Volume 0.01 mL

Reagent Volume 1.0 mL

Total Volume 1.01 mL

Low Normal 135 mg/dL

High Normal 200 mg/dL

Calibrator 200 mg/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE:

For instruments requiring a total volume of 1.0 mL, increase CHOLESTEROL LIQUID REAGENT volume to 2.0 mL and Sample volume to 0.02 mL. Proceed as outlined.

1. Dispense 1.0 mL CHOLESTOL LIQUID REAGENT into tubes labeled: Reagent Blank, Calibrator, Control, Sample 1, etc.

2. Prewarm for five minutes at 37º C.

3. Place 0.01 mL Specimen into appropriately labeled tube and mix well. Use D-H2O as sample for Reagent Blank.

4. Incubate all tubes at 37º C for 10 minutes.

5. Zero instrument at 520 nm using Reagent Blank.

6. Read and record absorbances for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out to concentration of Calibrator and read the Unknown concentration directly.

SERUM BLANK:

Highly lipemic Specimens require a Serum Blank.

1. Add 0.01 mL serum to 1.00 mL 0.9% sodium chloride solution.

2. Zero instrument at 520 nm with 0.9% sodium chloride solution.

3. Read and record absorbance of Serum Blank.

4. Subtract blank absorbance from sample test absorbance measured in Step 6 of PERFORMANCE OF TEST. Use this value in calculating the total cholesterol concentration.

STABILITY OF FINAL REACTION PRODUCT:

The test sample should be read within 30 minutes after color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-600 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use CHOLESTEROL CALIBRATOR (Cat. No. 2502) which is provided with the reagent set or other suitable calibration material for this purpose. Specimens exceeding 600 mg/dL should be diluted with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain the final answer.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for the Unknowns. EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera must be thoroughly investigated before patient results are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

Unknown (mg/dL) =

Unk. Abs.

---------------- X Cal. Conc. (mg/dL)

Cal. Abs.

EXAMPLE:

A 200 mg/dL Calibrator has Abs. = 0.328 while the Unknown Abs. = 0.265. The cholesterol concentration of the Unknkown is:

0.265           

---------- X       200 mg/dL   =   162 mg/dL

0.328

NOTE: To convert from mg/dL to SI units (mM/L), multiply the result in mg/dL by 10 dL/L and divide by 386.6 mg/mM.

LIMITATIONS

Several cholesterol analogs also react with cholesterol oxidase, but these substances appear in very small amounts in serum (4).

EXPECTED VALUES (6)

Recommended 200 mg/dL

Moderate 200 - 239 mg/dL

High Risk 240 mg/dL   

This range is taken from literature reference (6). Since cholesterol values vary with age, diet, exercise and geographic location (1), it is recommended that each laboratory establish its own range of expected values. Reference ranges based on an apparently healthy population are generally higher than the recommended (desirable) levels for adults. The EXPECTED VALUES as presented is the "recommended" (desirable) level for adults and the adult levels in term of risk for coronary heart disease.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This procedure is linear to 600 mg/dL.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to estalbish run to run precision.

WITHIN RUN

MEAN / ST. DEV. / %CV

Normal 135 / 1.3 / 1.0

Abnormal 335 / 2.9 / 1.0

RUN TO RUN

Normal 139 / 2.1 / 1.5

Abnormal 342 /  4.9 / 1.4

SPECIFICITY:

A comparison of this TOTAL CHOLESTEROL (LIQUID) PROCEDURE with another widely used commercial method showed a 99% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This TOTAL CHOLESTEROL (LIQUID) PROCEDURE has a sensitivity of 0.6 mg/dL per 0.001 absorbance unit.

REFERENCES

1. Tiez, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1982, p. 506.

2. Gordon, T., Castelli, W.P., Hjortland, M.C., Kannel, W.B., and Dawber T.R., Amer. J. Med., 62, 707 (1977).

3. World Health Organization Memorandum: Classification of Hyperlipidemias and Hyperlipoproteinemias. Circulation 45, 501 (1972).

4. Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., and FU, P.C., Clin.Chem. 20, 470 (1974).

5. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin. Chem., Vol. 21, p. 278D (1975).

6. The Expert Panel: Report of the National Cholesterol Education Program Expert Panel on detection, evaluation and treatment of high blood cholesterol in adults. Arch. Int. Med., 148: 36 - 39, 1988.