SUMMARY AND EXPLANATION

Castelli et al (1) have established an inverse relationship between serum high-density lipoprotein (HDL) cholesterol and the risk of coronary heart disease. Total and HDL cholesterols, in conjunction with a triglyceride determination, provide valuable information for the prediction of coronary heart disease (2).

The EAGLE POLIPOL PRECIPITATING REAGENT offers a means for rapid and complete separation of HDL from other serum lipoproteins. The isolated HDL fraction can be analyzed then for cholesterol content. The separation is based on a poly-alcohol precipitation of lower-density lipoproteins (LDL and VLDL) described by Izzo et al (3). The POLIPOL PRECIPITATING REAGENT does not contain metal ions and differs from first generation precipitating reagents that use divalent metal ions (e.g. Mn, Mg, Ca) to form complexes with LDL and VLDL. The molecular interactions involving metal ions are relatively weak and depend on time, temperature, ionic strength and metal binding agents. Furthermore, metal ions can interfere with some of the more commonly used cholesterol reagents (4).

PRINCIPLE

POLIPOL PRECIPITATING REAGENT interacts directly with LDL and VLDL at pH = 10 to form insoluble complexes. This action occurs at room temperature. The precipitate can be removed by centrifugation, and the supernatant is analyzed for HDL cholesterol.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2510 provides:

POLIPOL PRECIPITATING REAGENT - (Cat. No. 2511)

REACTIVE INGREDIENTS:

200 g/L polyethylene glycol buffered at pH = 10. Preservatives added.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity would indicate deterioration.

HDL CHOLESTEROL CALIBRATOR - (Cat. No. 2512)

REACTIVE INGREDIENTS:

0.65 mM cholesterol in alcohol. Equal to 50 mg/dL in the method.

PRECAUTIONS:

Do not ingest.

STORAGE AND STABILITY:

Store at 15 - 30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

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

Use a spectrophotometer or colorimeter calibrated at 520 nm and a Clinical Centrifuge capable of 1000 X g.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Unhemolyzed serum is recommended.

2. The Specimen need not be fasting.

SAMPLE STORAGE:

HDL cholesterol appears stable for 3 days at 2 - 30ºC.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Heparinized plasma should not be used. Gentisic acid significantly affects the enzymatic cholesterol assay. Young et al, (6) have reviewed drug effects on serum cholesterol.

PROCEDURE

MATERIALS PROVIDED:

POLIPOL PRECIPATING REAGENT (Cat. No. 2511) and HDL CHOLESTEROL CALIBRATOR (Cat. No. 2512).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.1 and 0.2 micropipettors

2. 1.0 mL pipettor or dispensor

3. Clinical centrifuge capable of 1000 X g

4. Incubator capable of maintaining 37ºC

5. CHOLESTEROL REAGENT SET (Cat. No. 2500)

6. Test tubes and rack

REACTION CONDITIONS:

SEPARATION:

Specimen Volume 0.2 mL

Polipol Reagent Vollume 0.2 mL

Incubation Temperature Ambient

Incubation Time 10 minutes

Centrifugation 10 minutes

CHOLESTEROL ASSAY:

Wavelength 520 nm

Filter Selection 500 - 540 nm

Reaction Type Endpoint

Reaction Time 10 minutes at 37º C

and Temperature 15 minutes at 30º C

0 minutes at 25º C

Sample Volume 0.1 mL

Reagent Volume 1.0 mL

Total Volume 1.1 mL

Low Normal 26 mg/dL

High Normal 75 mg/dL

Calibrator Value 50 mg/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

HDL SEPARATION

1. Dispense 0.2 mL Specimen into tubes labeled Control, Sample 1, etc.

2. Add 0.2 mL POLIPOL PRECIPITATING REAGENT. Mix well and allow to stand at ambient temperature for 10 minutes.

3. Centrifuge at 1000 X g (3/4 speed) for 10 minutes.

4. Make certain supernatant is clear before using in cholesterol assay. If the supernatant is cloudy, dilute the Specimen X 2 with D-H2O and repeat the separation. Multiply assay result X 2 to get final answer.

HDL CHOLESTEROL ASSAY

1. Dispense 1.0 mL CHOLESTEROL REAGENT into tubes labeled Reagent Blank, HDL Calibrator, Control, Sample 1, etc.

2. Pre-warm for 5 minutes at 37ºC.

3. Place 0.1 mL Sample into appropriately labeled tube and mix well. Use D-H2O as sample for Reagent Blank.

4. Incubate all trubes at 37ºC for 10 minutes.

5. Zero instrument at 520 nm using the Reagent Blank.

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

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

STABILITY OF FINAL REACTION PRODUCT:

The test samples 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 - 120 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use HDL CHOLESTEROL CALIBRATOR (Cat. No. 2512) which is provided in the reagent set or other suitable calibration material for this purpose.

QUALITY CONTROL:

The realiability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for 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 should be thoroughly investigated before patient values 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 50 mg/dL HDL Calibraotr had Abs.= 0.375 while the Unknown Abs.= 0.248. The HDL Cholesterol concentration of the Unknown is:

0.248 X 50 mg/dL

--------------------------  =   33 mg/dL

 0.375

NOTE: To convert 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

1. HDL cholesterol analysis is intended for the assessment of coronary heart disease risk in apparently healthy individual patients with hyperlipoproteinemis, liver disease or  recent myocardial infarction have no correlation.

2. Liebermann-Burchard cholesterol reagents can be used to assay HDL cholesterol, but sample reagent volumes should not be altered and sensitivity is limited.

EXPECTED VALUES

Males 26 - 63 mg/dL

Females 33 - 75 mg/dL

The ranges represents the 95% confidence intervals for a study of 33 males and 36 females from a clinically normal population. It is recommended that each laboratory establish its own range of expected values.

INTERPRETATION OF RESULTS

Serum HDL cholesterol is the single best indicator of coronary heart disease risk in an apparently normal person (2). Generally, HDL cholesterol levels below 35 mg/dL represent high risk, from 35 - 55 mg/dL an intermediate risk, and above 55 mg/dL a low riskA more accurate assessment can be established by considering the serum total cholesterol to HDL cholesterol ratio (5). These risk factors are sex-dependent.

RISK   TOTAL / HDL RATIO

MEN       WOMEN

1/2 average 3.4 3.3

average 5.0 4.4

2 X average 9.6 7.0

3 X average 24.0 11.0
 
 

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 120 mg/dL.

PRECISION:

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

WITHIN RUN

MEAN / ST. DEV. / %CV

Normal 48 / 0.9 / 1.9

Abnormal 23 / 0.6 / 2.6

RUN TO RUN

Normal 47 / 1.3 / 2.8

Abnormal 24 / 0.7 / 2.9

SPECIFICITY:

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

SENSITIVITY:

This HDL CHOLESTEROL PROCEDURE has a sensitivity of 0.8 mg/dL per 0.001 absorbance unit.

REFERENCES

1. Castelli, W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., Hulley, S.B., Kagen, A., and Zukel, W.J., Circulation 55, 787 (1977).

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

3. Izzo, C., Grillo, F., and Murador, E., Clin. Chem. 27, 371 (1981).

4. Liedtke, R.J., Busby, B., and Batjer, J.D., Clin. Chem. 24, 161 (1978).

5. Castelli, W.P., and Levitas, I.M., Current Prescribing 6, 39 (1977).

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