Laboratory

HDL Cholesterol:-Range, Precipitation and CHOD-PAP Method of Measurement

High-density lipoproteins (HDL) are the types of lipoproteins that carry cholesterol from the peripheral tissues to the liver. In the liver, excretion of cholesterol takes place. Thus, HDL cholesterol is considered good cholesterol. That means the greater value of HDL, reduces the chances of heart diseases. You can measure the amount of HDL cholesterol in the serum by precipitation method followed by CHOD-PAP method.

HDL Cholesterol Range

The table shown below is the level of HDL cholesterol found in the bloodstream (serum). However, this is an approximate value. This value can slightly vary from one laboratory to another, based on the method of testing, the type of reagent used, etc. It can also differ due to geographical variation, habitat, age, sex, etc.

No risk Moderate risk Risk
Men > 55 mg/dl 35-55 mg/dl < 35 mg/dl
Women >  65 mg/dl 45- 65 mg/dl < 45 mg/dl

If the level of HDL cholesterol in the blood becomes abnormal, he/she should visit the doctor for further investigation.

How to Increase HDL Cholesterol?

Since HDL cholesterol is good cholesterol, it is better to increase its level in the blood. Some of the ways to increase its level in the blood are stated below.

  • Do regular physical exercises. This will help you to reduce your excess body weight.
  • Reduce the consumption of unhealthy fat of meat, butter, cheese, etc. Avoid processed foods. Instead of that, consume unsaturated fats from vegetable oils, avocado, etc.
  • Avoid alcoholism because it never does you any good.
  • Avoid smoking or secondhand smoking.
  • Reduce the excess consumption of carbohydrates, sugar, etc from your diet.

Precipitation Method+  CHOD-PAP Method for HDL Cholesterol Measurement

Principle Reaction

When the serum reacted with the precipitation reagent and then centrifuged, the HDL will remain as a supernatant. Low-density lipoproteins( LDL), and very-low-density lipoproteins (VLDL) will precipitate out.

After that, HDL is assayed with the help of the cholesterol (CHOD/PAP) reagent.

Sample Preparation

The table, shown below for HDL cholesterol measurement is valid for the reagents of Tulip, Diasys, and the Analyticon Companies.  So, if you have the reagent kit of any other company, then there can be minor fluctuation in the method.

HDL-cholesterol, Precipitation Method
Precipitation Method

Mix sample with the precipitating reagent. Then incubate the mixture at room temperature for about 10 mins. Finally, centrifuge it at 4000 rpm for 10 mins to obtain the clear supernatant.

If you have two reagents, then you can make a working solution out of it. Go, through your literature to know the composition. Else, a single reagent can work as a working solution.

After obtaining a clear supernatant solution from the above process, mix it with the CHOL reagent which follows the CHOD-PAP method. Then incubate the solution at 37°C for 5 mins in a water/dry bath. After that feed the solution into a semi-auto biochemistry analyzer/colorimeter.

An unclear supernatant can be an indication of the incomplete precipitation of lipoproteins or part of the precipitate is floating on the surface. In both cases, there will be a high concentration of triglycerides (TG). A high TG value (above 1000 mg/dl) will give you an error in your measurement. In that case, again dilute the precipitate solution with distilled water or 0.9%  NaCl at a ratio of 1: y. Repeat the steps from the beginning. Finally, when you obtain the value of HDL cholesterol, multiply it with the dilution factor (1+y).

For making a reagent blank replace the supernatant with the same volume of distilled water from the above method.

Always remember that sipping volume < sample volume + reagent volume. So that means, make sufficient sample volume to feed into the semi-auto biochemistry analyzer. Else, it will suck up the air and will give the fault reading.

3. Programming of HDL Cholesterol for a Semi-auto Biochemistry Analyzer (Precipitation Method + CHOD-PAP Method)

HDL-cholesterol, precipitation method, CHOD-PAP method
Precipitation and CHOD-PAP Method

4. Handling of HDL Cholesterol Reagent

  • Wear an apron and surgical gloves before carrying out the measurement.
  • Look for the expiry date of the reagents during purchase and measurement time. Suppliers tend to give you reagent kits with a low expiry interval.
  • Once you open the reagent bottles, the lifespan will decrease. So, look in your reagent kit literature for that.
  • Bring the reagents and samples to room temperature before you can carry out any measurement.
  • Always store reagents in the refrigerator when not in use.

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